Nucleotide sequences involved in plant disease resistance

ABSTRACT

The present invention relates to methods for producing plants having enhanced disease resistance. NRC1 proteins and nucleic acid sequences encoding these are provided, as well as transgenic plants producing NRC1 proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present patent application is a continuation-in-part of U.S.application Ser. No. 10/481,335, filed Jun. 17, 2004, which is anational phase application under 35 U.S.C. § 371 of PCT/NL02/00417(published as WO03/000930), filed Jun. 24, 2002, which claims priorityto EP01202420.4, filed Jun. 22, 2001, each of which is incorporated byreference its entirety for any purpose.

FIELD OF THE INVENTION

The present invention relates to transgenic plants and plant cellscomprising a gene encoding an NRC1 protein (NB-LRR Required forHR-associated Cell Death 1) integrated in its genome and methods formaking such plants and cells. Especially Solanaceae plants and plantparts (seeds, fruit, leaves, etc.) with enhanced disease resistance areprovided. Also provided are isolated nucleic acid molecules encodingNRC1 proteins according to the invention, vectors comprising these, aswell as isolated NRC1 proteins themselves. Further, plant cells andplants comprising one or more mutations in an endogenous NRC1 allele areprovided, whereby the mutation(s) confer enhanced diseases resistance tothe plants and plant cells.

BACKGROUND OF THE INVENTION

Active defense of plants, triggered upon recognition of an avirulencefactor of a pathogen mediated by a resistance gene, follows thegene-for-gene model (Dangl and Jones, 2001, Nature 411, 826-833). Todate, several plant resistance genes (R genes) have been cloned andbased on the structure of the proteins they encode, the genes aredivided into several groups (Hammond-Kosack and Jones, 1997, Annu. Rev.Plant Physiol. Plant Molec. Biol. 48, 575-607). Most R genes encodecytoplasmic NB-LRR proteins, containing a nucleotide binding site (NB)and leucine-rich repeats (LRR). This group consists of genes encodingCC-NB-LRR proteins, containing a coiled-coil domain and genes thatencode proteins that have a domain similar to mammalian Toll andinterleukin (IL) receptors, the so-called TIR-NB-LRR proteins(Hammond-Kosack and Jones, 1997, supra).

Using such specific resistance genes in breeding programs for durableresistance is problematic since pathogens easily circumvent recognitionby mutations in their avirulence factors, thereby preventing inductionof active defense (Westerink et al., 2004, Mol. Microbiol. 54, 533-545).Similarity among resistance proteins (R proteins) suggests the existenceof common resistance pathways (Shirasu and Schulze-Lefert, 2000, PlantMol. Biol. 44, 371-385). Therefore, identification of additional genesrequired for resistance not only provides information on how suchsignaling pathways function but might also enable us to identify genesthat play a more general role in resistance. For example, byvirus-induced gene silencing (VIGS) in Nicotiana benthamiana it wasshown that SGT1 is involved in multiple defense pathways, such as N-,Rx- and Pto-mediated HR and resistance, and Cf-4- and Cf-9-mediated HR(Peart et al., 2002, Proc. Natl. Acad. Sci. USA 99, 10865-10869; Zhanget al., 2004, Plant J. 40, 213-224). SGT1 is an interactor of SKP1,which is a component of the SCF E3-ligase complex that is involved inubiquitination of proteins, a modification which targets them fordegradation (Schwechheimer and Schwager, 2004, Plant Cell Reports 23,353-364). It is hypothesized that silencing an essential gene of thisprotein degradation system hampers the ubiquitination process, therebyinhibiting the degradation of negative regulators, which is required fordefense activation (Azevedo et al., 2002, Science 295, 2073-2076).

In several resistance pathways MAPKs (mitogen activated protein kinases)are activated (Zhang and Klessig, 2001, Trends Plant Sci. 6, 520-527;Pedley and Martin, 2005, Curr. Opin. Plant Biol. 8, 541-547). InCf-9-containing tobacco plants and cell cultures challenged with Avr9,NtWIPK (wound-induced protein kinase) and NtSIPK (salicylic acid-inducedprotein kinase) are activated (Romeis et al., 1999, Plant Cell 11,273-287). VIGS of a NtCDPK (calcium-dependent protein kinase) in N.benthamiana inhibits the Cf-9/Avr9- and Cf-4/Avr4-dependent HR (Romeiset al., 2001, EMBO J. 20, 5556-5567) and VIGS of LeACIK1 (Avr/Cf-inducedkinase 1) in tomato results in decreased C. fulvum resistance (Rowlandet al., 2005, Plant Cell 17, 295-310). The activation of kinases duringdefense and the decreased resistance upon ‘knock-down’ of their encodinggenes supports their function in defense activation.

Following a biased approach, 21 genes known to be involved indefense-related signaling were used for VIGS in tomato and it was foundthat nine of them are involved in Pto-mediated resistance. Among theseare two genes encoding MAPKKs (LeMEK1 and LeMEK2) and two genes encodingMAPKs (LeNTF6 and LeWIPK) (Ekengren et al., 2003, Plant J. 36, 905-917).In another study, over 2400 cDNAs from a normalized library of N.benthamiana cDNA were cloned in a Potato Virus X-based vector and usedfor VIGS in N. benthamiana. About 3% of the cDNAs affected Pto-dependentHR upon silencing. Among these a MAPKKKα was identified as a positiveregulator of both resistance and disease (Del Pozo et al., 2004, EMBO J.23, 3072-3082).

Lu et al. (2003, EMBO J. 22, 5690-5699) performed VIGS using 4992 cDNAsfrom a normalized N. benthamiana cDNA library cloned into a PVX vector.Of the cDNAs, 79 (1.6%) corresponded to genes required for Pto-mediatedHR, whereas silencing of only six of them also impaired Pto-mediatedresistance against Pseudomonas syringae. VIGS using a cDNA correspondingto HSP90 abolished not only Pto-mediated HR but also Pto-, Rx- andN-mediated resistance, indicating that HSP90 is required in multipledisease resistance pathways. The same set of cDNAs was also used forVIGS in N-transgenic N. benthamiana, after which the plants wereinoculated with a GFP-tagged strain of TMV. Resistance against TMV wasmost significantly suppressed upon silencing using a cDNA fragmentderived from a CC-NB-LRR-encoding gene, referred to as NRG1 (Nrequirement gene 1) (Peart et al., 2005, Curr. Biol. 15, 968-973). NRG1was shown to be specifically required for N gene function, indicatingthat CC-NB-LRR proteins do not only act as resistance proteins involvedin recognition of avirulence factors, but are also involved in thesignaling pathway initiated by the TIR-NB-LRR protein N, whicheventually leads to resistance (Peart et al., 2005, supra). Thus,although the tobacco NRG1 protein functions downstream of the plant'sdefense signaling cascade initiated by a resistance protein, it has thedrawbacks that it is specifically involved in N-mediated resistanceagainst tobacco mosaic virus (TMV) and is not a general cofactor ofdisease resistance (Rx- and Pto-mediated resistance against PVX andPseudomonas syringae were unaffected by NRG1 silencing), whereby it maynot be suitable for creating broad pathogen resistance in crops such astomato.

Despite the increasing information about disease resistance pathways,there is still a need in identifying genes and proteins which can beused to create plants with durable, broad range disease resistance. Itis an object of the invention to provide such nucleic acids, proteinsand methods for creating plants, especially plants belonging to thefamily Solanaceae, with enhanced disease resistance.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for producing a transgenic planthaving enhanced disease resistance compared to a non-transgenic controlplant. In some embodiments, the methods comprise the steps of:

-   (a) transforming a plant or plant cell with a nucleotide sequence    encoding an NRC1 protein operably linked to a promoter active in    plant cells,-   (b) regenerating a plant.

In some embodiments, said nucleotide sequence is integrated into thegenome of said plant.

In some embodiments, the methods further comprise:

-   (c) screening the regenerated plant, or a plant derived therefrom by    selfing or crossing, for resistance to one or more plant pathogens    and identifying a plant comprising enhanced resistance to one or    more of said plant pathogens.

In some embodiments, said promoter is a pathogen inducible promoter.

In some embodiments, the NRC1 protein comprises the amino acid sequenceof SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising atleast 70% amino acid identity to SEQ ID NO: 2 over its entire length.

In some embodiments, the plant belongs to the family Solanaceae. In someembodiments, the plant is of the genus Solanum.

The present invention also provides transgenic plants, plant cells,seeds or fruits, obtainable by the methods described herein.

The present invention also provides plants, plant cells, seeds or fruitscomprising a chimeric gene, the chimeric gene comprising a promoteractive in plant cells operably linked to a nucleic acid encoding SEQ IDNO: 2 or SEQ ID NO: 4, or an amino acid sequence comprising at least 70%amino acid sequence identity to SEQ ID NO: 2 over the entire length. Insome embodiments, the plant is of the genus Solanum.

The present invention also provides isolated proteins comprising theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acidsequence comprising at least 70% amino acid sequence identity to SEQ IDNO: 2 over the entire length.

The present invention also provides isolated nucleic acid moleculesencoding a protein comprising the amino acid sequence of SEQ ID NO: 2 orSEQ ID NO: 4, or an amino acid sequence comprising at least 70% aminoacid sequence identity to SEQ ID NO: 2 over the entire length.

The present invention also provides a chimeric gene comprising apromoter active in plant cells operably linked to a nucleic acidmolecule according to encoding a protein comprising the amino acidsequence of SEQ ID NO: 2 or SEQ ID NO: 4, or an amino acid sequencecomprising at least 70% amino acid sequence identity to SEQ ID NO: 2over the entire length. In some embodiments, 3′ untranslated nucleicacid molecule is operably linked to the chimeric gene.

The present invention also provides vectors comprising the chimeric genedescribed above.

GENERAL DEFINITIONS

“HR” refers to the hypersensitive response, i.e. local plant cell death,seen as either microscopic lesions (as described by Rivas and Thomas,2005, Ann Rev Phytopath 43: 395-436) and/or macroscopic lesions.Hypersensitive cell death is usually associated with other plantresponses, such as production of reactive oxygen species and theactivation of defense related genes in cells surrounding the HR lesion.

“Plant pathogens” refer to biotic agents which are capable of causingdisease on plants, such as plant pathogenic fungi, bacteria, viruses,oomycetes, mycoplasma like organisms, nematodes, white fly and aphidsand the like. Generally all strains, races or pathovars of a pathogenspecies which are capable of causing disease on host tissue are includedherein.

“Biotrophic plant pathogens” or “biotroph” refers to a pathogen thatkeeps the host plant cells alive and relies on living cells for growthand tissue colonization.

“Hemibiotrophic plant pathogen” or “hemibiotroph” refers to a plantpathogen which keeps the host cells alive during at least part of itslife cycle.

“Necrotrophic plant pathogen” refers to a plant pathogen which activelykills plant cells upon tissue colonization, by producing toxic enzymes,proteins or metabolites that kill host cells.

“Elicitor independent HR” refers to a hypersensitive response whichdevelops without a pathogen or a pathogen elicitor (e.g. a fungal Avrprotein) being present.

When referring to plants expressing an NRC1 protein according to theinvention (e.g. a constitutively active NRC1 protein) one may alsodistinguish between “constitutive HR”, whereby reference is made to thedevelopment of HR lesions in the absence of pathogens or pathogenelicitor proteins, and “induced HR”, whereby reference is made to thedevelopment of HR lesions following the presence of an inducing stimulus(e.g. following induction of the promoter which drives expression of thenucleic acid sequence encoding the NRC1 protein, or variant thereof).

“Solanaceae” refers herein to plant genera, species, and varietiesthereof, belonging to the family Solanaceae. These include speciesbelonging to the genus Solanum (including Solanum lycopersicum, whichused to be known as Lycopersicon esculentum), Nicotiana, Capsicum,Petunia and other genera.

“Disease resistance” refers herein to various levels of diseaseresistance or tolerance of a plant, including moderate resistance andhigh resistance or complete resistance to one or more pathogens. It canbe measured and optionally quantified by comparison of pathogen causedsymptoms (such as frequency and/or size of HR lesions, fungal mycelium,etc.) relative to those seen in susceptible control plants when grownunder identical disease pressure. Such disease bioassays can be carriedout using known methods. Disease resistance can also be indirectlymeasured as higher yield of resistant plants compared to susceptibleplants when grown under disease pressure.

“Enhanced disease resistance” refers to any statistically significantincrease in disease resistance of a plant or plant tissue compared to asuitable control. Both a qualitative increase (e.g. from susceptible toresistant) and a quantitative increase are encompassed herein. Alsoencompassed is both a reduction of disease incidence (percentage ofplants becoming infected) and/or of disease severity. Preferably, aplant having enhanced disease resistance to at least one pathogen is aplant comprising at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%,90%, or even 100% higher levels of resistance to the pathogen than thecontrol plant, using appropriate bioassays and/or field assays forassessing disease resistance.

“Broad spectrum” disease resistance refers to enhanced resistanceagainst at least two, three, four, or more pathogens of differentpathogen species. For example, a host plant having enhanced resistanceto several biotrophic and/or hemibiotrophic and/or necrotrophic pathogenspecies would be considered to have broad spectrum resistance.

“Pathogen caused symptoms” include any symptoms of disease, such asmycelium growth/biomass on/in the host tissue, bacterial growth/biomass,size and/or frequency of necrotic or chlorotic lesions on plant tissue,size and/or frequency of cankers, etc.

The term “nucleic acid sequence” (or nucleic acid molecule) refers to aDNA or RNA molecule in single or double stranded form, particularly aDNA encoding a protein or protein fragment according to the invention.An “isolated nucleic acid sequence” refers to a nucleic acid sequencewhich is no longer in the natural environment from which it wasisolated, e.g. the nucleic acid sequence in a bacterial host cell or inthe plant nuclear or plastid genome.

The terms “protein” or “polypeptide” are used interchangeably and referto molecules consisting of a chain of amino acids, without reference toa specific mode of action, size, 3 dimensional structure or origin. A“fragment” or “portion” of a protein may thus still be referred to as a“protein”. An “isolated protein” is used to refer to a protein which isno longer in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell.

“Functional”, in relation to NRC1 proteins (or variants, such asorthologs or mutants, and fragments), refers to the capability to modifythe (quantitative and/or qualitative) development of HR lesions and/orthe level of disease resistance by modifying the expression level of theNRC1-encoding gene (e.g. by overexpression or silencing) in a plant. Forexample, the functionality of a putative NRC1 protein obtained fromplant species X can be tested by various methods. If the protein isfunctional, silencing of the NRC1 gene encoding the protein in plantspecies X, using e.g. VIGS or gene silencing vectors, will lead to areduction or suppression of pathogen- or elicitor induced HR lesionsand/or a reduction of pathogen resistance, as shown in the Examples fortomato. Also, complementation with a functional NRC1 protein will becapable of restoring HR lesions and/or pathogen resistance.Alternatively, transient or stable (over)expression in species X of thegene encoding the NRC1 protein (optionally together with aposttranscriptional gene silencing inhibitor) will lead to thedevelopment of elicitor independent HR lesions and/or enhanced diseaseresistance, especially against biotrophic and/or hemi-biotrophicpathogens. See also the Examples.

The term “gene” means a DNA sequence comprising a region (transcribedregion), which is transcribed into an RNA molecule (e.g. an MRNA) in acell, operably linked to suitable regulatory regions (e.g. a promoter).A gene may thus comprise several operably linked sequences, such as apromoter, a 5′ leader sequence comprising e.g. sequences involved intranslation initiation, a (protein) coding region (cDNA or genomic DNA)and a 3′ non-translated sequence comprising e.g. transcriptiontermination sites.

A “chimeric gene” (or recombinant gene) refers to any gene, which is notnormally found in nature in a species, in particular a gene in which oneor more parts of the nucleic acid sequence are present that are notassociated with each other in nature. For example the promoter is notassociated in nature with part or all of the transcribed region or withanother regulatory region. The term “chimeric gene” is understood toinclude expression constructs in which a promoter or transcriptionregulatory sequence is operably linked to one or more coding sequencesor to an antisense (reverse complement of the sense strand) or invertedrepeat sequence (sense and antisense, whereby the RNA transcript formsdouble stranded RNA upon transcription).

“3′ UTR” or “3′ non-translated sequence” (also often referred to as 3′untranslated region, or 3′ end) refers to the nucleic acid sequencefound downstream of the coding sequence of a gene, which comprises forexample a transcription termination site and (in most, but not alleukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA orvariants thereof). After termination of transcription, the mRNAtranscript may be cleaved downstream of the polyadenylation signal and apoly(A) tail may be added, which is involved in the transport of themRNA to the cytoplasm (where translation takes place).

“Expression of a gene” refers to the process wherein a DNA region, whichis operably linked to appropriate regulatory regions, particularly apromoter, is transcribed into an RNA, which is biologically active, i.e.which is capable of being translated into a biologically active proteinor peptide (or active peptide fragment) or which is active itself (e.g.in posttranscriptional gene silencing or RNAi). An active protein incertain embodiments refers to a protein being constitutively active. Thecoding sequence is preferably in sense-orientation and encodes adesired, biologically active protein or peptide, or an active peptidefragment. In gene silencing approaches, the DNA sequence is preferablypresent in the form of an antisense DNA or an inverted repeat DNA,comprising a short sequence of the target gene in antisense or in senseand antisense orientation. “Ectopic expression” refers to expression ina tissue in which the gene is normally not expressed.

A “transcription regulatory sequence” is herein defined as a nucleicacid sequence that is capable of regulating the rate of transcription ofa (coding) sequence operably linked to the transcription regulatorysequence. A transcription regulatory sequence as herein defined willthus comprise all of the sequence elements necessary for initiation oftranscription (promoter elements), for maintaining and for regulatingtranscription, including e.g. attenuators or enhancers. Although mostlythe upstream (5′) transcription regulatory sequences of a codingsequence are referred to, regulatory sequences found downstream (3′) ofa coding sequence are also encompassed by this definition.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one of skill in the art to act directly orindirectly to regulate the amount of transcription from the promoter. A“constitutive” promoter is a promoter that is active in most tissuesunder most physiological and developmental conditions. An “inducible”promoter is a promoter that is physiologically (e.g. by externalapplication of certain compounds) or developmentally regulated. A“tissue specific” promoter is only active in specific types of tissuesor cells. A “promoter active in plants or plant cells” refers to thegeneral capability of the promoter to drive transcription within a plantor plant cell. It does not make any implications about thespatiotemporal activity of the promoter.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter, or rather atranscription regulatory sequence, is operably linked to a codingsequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein encoding regions,contiguous and in reading frame so as to produce a “chimeric protein”. A“chimeric protein” or “hybrid protein” is a protein composed of variousprotein “domains” (or motifs) which is not found as such in nature butwhich a joined to form a functional protein, which displays thefunctionality of the joined domains (for example a Coiled Coil domain(CC), a nucleotide binding domain (NB-ARC) and a Leucine Rich Repeat(LRR) region may be combined). A chimeric protein may also be a fusionprotein of two or more proteins occurring in nature. The term “domain”as used herein means any part(s) or domain(s) of the protein with aspecific structure or function that can be transferred to anotherprotein for providing a new hybrid protein with at least the functionalcharacteristic of the domain. Specific domains can also be used toidentify other NRC1 proteins, such as NRC1 orthologs from other plantspecies.

The terms “target peptide” refers to amino acid sequences which target aprotein, or protein fragment, to intracellular organelles such asplastids, preferably chloroplasts, mitochondria, or to the extracellularspace or apoplast (secretion signal peptide). A nucleic acid sequenceencoding a target peptide may be fused (in frame) to the nucleic acidsequence encoding the amino terminal end (N-terminal end) of the proteinor protein fragment, or may be used to replace a native targetingpeptide.

A “nucleic acid construct” or “vector” is herein understood to mean aman-made nucleic acid molecule resulting from the use of recombinant DNAtechnology and which is used to deliver exogenous DNA into a host cell.The vector backbone may for example be a binary or superbinary vector(see e.g. U.S. Pat. No. 5,591,616, US 2002138879 and WO95/06722), aco-integrate vector or a T-DNA vector, as known in the art and asdescribed elsewhere herein, into which a chimeric gene is integrated or,if a suitable transcription regulatory sequence is already present, onlya desired nucleic acid sequence (e.g. a coding sequence, an antisense oran inverted repeat sequence) is integrated downstream of thetranscription regulatory sequence. Vectors usually comprise furthergenetic elements to facilitate their use in molecular cloning, such ase.g. selectable markers, multiple cloning sites and the like (seebelow).

A “host cell” or a “recombinant host cell” or “transformed cell” areterms referring to a new individual cell (or organism) arising as aresult of at least one nucleic acid molecule, especially comprising achimeric gene encoding a desired protein or a nucleic acid sequencewhich upon transcription yields an antisense RNA or an inverted repeatRNA (or hairpin RNA) for silencing of a target gene/gene family, havingbeen introduced into said cell. The host cell is preferably a plant cellor a bacterial cell. The host cell may contain the nucleic acidconstruct as an extra-chromosomally (episomal) replicating molecule, ormore preferably, comprises the chimeric gene integrated in the nuclearor plastid genome of the host cell. Throughout the text the term “host”may also refer to the host plant species which a pathogen is able toinvade or infect, but this will be clear from the context. Plant speciesare classified as “host” or “non-host” species in relation to apathogen. “Non-host” species are completely immune to pathogen infectionof all races or strains of a pathogen, even under optimum conditions fordisease development. The “host” species are also referred to as the“host range” of a pathogen and are immune to certain (but not all) racesof a pathogen.

The term “selectable marker” is a term familiar to one of ordinary skillin the art and is used herein to describe any genetic entity which, whenexpressed, can be used to select for a cell or cells containing theselectable marker. Selectable marker gene products confer for exampleantibiotic resistance, or more preferably, herbicide resistance oranother selectable trait such as a phenotypic trait (e.g. a change inpigmentation) or a nutritional requirements. The term “reporter” ismainly used to refer to visible markers, such as green fluorescentprotein (GFP), eGFP, luciferase, GUS and the like.

The term “ortholog” of a gene or protein refers herein to the homologousgene or protein found in another species, which has the same function asthe gene or protein, but (usually) diverged in sequence from the timepoint on when the species harbouring the genes diverged (i.e. the genesevolved from a common ancestor by speciation). Orthologs of the tomatonrcl1 gene may thus be identified in other plant species based on bothsequence comparisons (e.g. based on percentages sequence identity overthe entire sequence or over specific domains) and functional analysis.

The terms “homologous” and “heterologous” refer to the relationshipbetween a nucleic acid or amino acid sequence and its host cell ororganism, especially in the context of transgenic organisms. Ahomologous sequence is thus naturally found in the host species (e.g. atomato plant transformed with a tomato gene), while a heterologoussequence is not naturally found in the host cell (e.g. a tomato planttransformed with a sequence from potato plants). Depending on thecontext, the term “homolog” or “homologous” may alternatively refer tosequences which are descendent from a common ancestral sequence (e.g.they may be orthologs).

“Stringent hybridisation conditions” can be used to identify nucleotidesequences, which are substantially identical to a given nucleotidesequence. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequences at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridises to a perfectly matchedprobe. Typically stringent conditions will be chosen in which the saltconcentration is about 0.02 molar at pH 7 and the temperature is atleast 60° C. Lowering the salt concentration and/or increasing thetemperature increases stringency. Stringent conditions for RNA-DNAhybridisations (Northern blots using a probe of e.g. 100 nt) are forexample those which include at least one wash in 0.2×SSC at 63° C. for20 min, or equivalent conditions. Stringent conditions for DNA-DNAhybridisation (Southern blots using a probe of e.g. 100 nt) are forexample those which include at least one wash (usually 2) in 0.2×SSC ata temperature of at least 50° C., usually about 55° C., for 20 min, orequivalent conditions. See also Sambrook et al. (1989) and Sambrook andRussell (2001).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they (whenoptimally aligned by for example the programs GAP or BESTFIT usingdefault parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength, maximizing the number of matches and minimises the number ofgaps. Generally, the GAP default parameters are used, with a gapcreation penalty=50(nucleotides)/8(proteins) and gap extensionpenalty=3(nucleotides)/2(proteins). For nucleotides the default scoringmatrix used is nwsgapdna and for proteins the default scoring matrix isBlosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequencealignments and scores for percentage sequence identity may be determinedusing computer programs, such as the GCG Wisconsin Package, Version10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego,Calif. 92121-3752 USA, or EmbossWin version 2.10.0 (using the program“needle”). Alternatively percent similarity or identity may bedetermined by searching against databases, using algorithms such asFASTA, BLAST, etc.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”. It is further understood that,when referring to “sequences” herein, generally the actual physicalmolecules with a certain sequence of subunits (e.g. amino acids) arereferred to.

As used herein, the term “plant” includes plant cells, plant tissues ororgans, plant protoplasts, plant cell tissue cultures from which plantscan be regenerated, plant calli, plant cell clumps, and plant cells thatare intact in plants, or parts of plants, such as embryos, pollen,ovules, fruit (e.g. harvested tomatoes), flowers, leaves, seeds, roots,root tips and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Predicted Sequence of the NRC1 Protein

The predicted sequence of the NRC1 protein (SEQ ID NO:2) is displayed.The first 150 amino acid residues represent the coiled-coil (CC) domainand residues that are predicted to form the CC structure are underlined.Residues 151 to 508 comprise the nucleotide-binding (NB-ARC) domain,with the following motifs (underlined and labeled): Kinase1A (P loop),RNBS-A, Kinase 2, RNBS-B, RNBS-C, GLPL, RNBS-D and MHD. Residues 509 to846 comprise the 13 imperfect leucine-rich repeats (LRRs); the conservedhydrophobic and proline residues are shown in bold. Below the proteinsequence the LRR consensus motif is indicated: ‘1’ indicates a conservedaliphatic residue, ‘c’ indicates a conserved charged residue and ‘P’indicates a conserved proline residue.

FIG. 2A—NRC1 is Required for Full Cf-4-Mediated HR of Tomato toCladosporium fulvum

Cf0 tomato and Cf-4-containing tomato plants were inoculated with theindicated TRV constructs and plants were analyzed three weeks after theonset of VIGS. Leaflets of TRV-infected Cf-4-containing tomato plantswere injected with Avr4 protein and examined for the development of anHR. The number of sites mounting an HR on TRV:00-infected plants was setto 100%. Each error bar represents the standard error from fourindependent experiments.

FIG. 2B—NRC1 is Required for Full Cf-4-Mediated Resistance of Tomato toCladosporium fulvum

Non-TRV infected and TRV-infected Cf-4 or Cf-0 plants were inoculatedwith C. fulvum-pGPD::GUS and two weeks post inoculation colonization ofthe leaflets was studied with an X-gluc assay.

FIG. 3—Inoculation of N. benthamiana with TRV:NRC1 Affects Cf/Avr-,LeEix2/tvEix-, Pto/AvrPto-and Rx/CP-Induced HR

N. benthamiana was inoculated with TRV:00 (empty vector), TRV:NRC1 andTRV:SGT1. Three weeks later leaves were infiltrated with Agrobacteriaexpressing HR-inducing proteins and pictures were taken at 4 days postinfiltration. First, second and third column: leaves of N. benthamianaexpressing the Cf-4 resistance gene agroinfiltrated with Avr4 or a mixof Cf-9 and Avr9, or a mix of LeEix2 and tvEix (combined in a 1:1ratio), respectively. Fourth column: leaves of transgenic N. benthamianaexpressing the Pto resistance gene agroinfiltrated with AvrPto. Fifthcolumn: leaves of transgenic N. benthamiana expressing the Rx resistancegene agroinfiltrated with the gene expressing the coat protein of PVX(CP). The dark circles indicate an HR, light circles indicate acompromised HR.

FIG. 4—Constitutively Active NRC1 Induces an Elicitor-Independent HR andAllows to Position NRC1 in a Cell Death Signaling Pathway

N. benthamiana expressing the Cf-4 resistance gene was agroinfiltratedwith the indicated genes. For panels A and C, three weeks prior toagroinfiltration the plants were inoculated with the indicated TRVconstructs. Dark circles indicate an HR, light circles indicate acompromised HR.

(A) Agroinfiltration of genes encoding constitutively active MAPKK andMAPK kinases. First column: agroinfiltration with the gene encoding theconstitutively active kinase domain of LeMAPKKKα (MAPKKK-KD). Secondcolumn: agroinfiltration with the gene encoding a constitutively activeform of LeMEK2 (MEK2DD). Two days post infiltration of MAPKKK-KD orMEK2DD expression was induced by spraying the leaves with estradiol.Pictures were taken four days post agroinfiltration.

(B) Agroinfiltration of wild-type NRC1 (wt) and mutated forms of thegene, under control of the 35S-promoter, either mixed in a 1:1 ratiowith Agrobacterium directing expression of the gene encoding silencingsuppressor p19 (left panel), or alone (right panel). NRC1^(K191R)(K191R): inactive P-loop mutant of NRC1; NRC1^(D481V) (D481V):constitutively active NRC1 (mutated in the MHD motif);NRC1^(K191R/D481V) (K191R/D481V): double mutant of NRC1. Pictures weretaken three days post agroinfiltration.

(C) Agroinfiltration of Avr4 and the gene encoding constitutively activeNRC1^(D481V) (D481V). Pictures were taken three days postagroinfiltration.

FIG. 5—Model for NRC1 Mediated Cell Death Signaling

Model based on epistasis experiments combining cell death assays andVIGS in N. benthamiana. Cf-4/Avr4 mediated cell death signals in anEDS1-, NRC1-, MEK2-, and SGT1/RAR1 dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have used cDNA-AFLP analysis, in combination withVIGS (Virus Induced Gene Silencing), to identify genes involved inCf-4/Avr4-dependent HR and disease resistance. Among the genes of whichVIGS resulted in a suppression of the Avr4-induced HR, one tomato genewas identified (referred herein to as NRC1), encoding a CC-NB-LRR typeresistance protein analog (herein referred to as NRC1 for NB-LRR proteinRequired for HR-associated Cell death 1). Silencing of NRC1 in tomatocompromised not only the development of an Avr4 induced HR, but alsoresistance to the tomato pathogen Cladosporium fulvum. This indicatedthat the tomato Cf-4 resistance protein (an extracellular receptor likeprotein) requires a cytoplasmic NB-LRR protein to be functional.

Furthermore, it was surprisingly found that NRC1 is involved in multipleHR and multiple disease resistance/cell death signaling pathways, suchas Cf-9/Avr-9-, LeEix2/Eix-, Pto/AvrPto- and Rx/CP-initiated HR (seeExamples). Further tests are being conducted to determine whether NRC1is also involved in other HRs, such as the Mi-mediated HR (conferringresistance to nematodes, white fly and aphid-induced HR; see U.S. Pat.No. 6,613,962 and EP0937155B1). Thus, NRC1 is involved in HR pathwaystriggered by both extra- and intracellular disease resistance proteinswhich belong to different classes: extracellular receptor like proteins(RLPs, such as Cf-4, Cf-9 and LeEix2), Ser/Thr protein kinases such asPto and a CC-NB-LRR protein (Rx), which confer resistance torespectively fungi (Cladosporium fulvum and Trichoderma viride), abacterium (Pseudomonas syringae pv tomato) or a virus (PVX).

The NRC1 protein (and the NRC1 gene encoding it) can be used to conferor enhance plant resistance against a variety of pathogens, especiallybiotrophic and hemi-biotrophic plant pathogens, but also necrotrophicplant pathogens such as Botrytis species. Especially, expression of NRC1(or variants or fragments thereof, as defined elsewhere) leads toenhanced resistance, especially against pathogens biotrophic and/orhemibiotrophic pathogens, i.e. all pathogens which obtain nutrients fromliving cells. Without limiting the scope of the invention, it is thoughtthat the knock-down (gene silencing) or knock-out (e.g. by TILLING) ofendogenous NRC1 genes can be used to confer or enhance resistanceagainst necrotrophic pathogens, as the pathway leading to necrosis isaffected and necrotophic pathogens require this pathway. Thus, dependingon the pathogen(s) against which resistance is to be enhanced, either anincrease or a decrease in NRC1 expression levels may be used to enhanceresistance. Optionally both approaches may be used in one plant, e.g.under control of different promoters. For example, NRC1 can be expressedunder control of a promoter induced by a (hemi)-biotrophic pathogen, toconfer resistance to biotrophic and/or hemibiotrophic leaf pathogens,while at the same time endogenous NRC1 gene (or gene family) can besilenced in certain tissues, or upon induction by a necrotroph using apromoter which is inducible by necrotrophic pathogens or wounding.

It was further found that, when a constitutively active NRC1 protein(NRC1^(D481V)) was produced transiently in tomato, the plant tissueshowed elicitor independent cell death (HR), showing that expression ofa functional NRC1 protein can be used to confer or enhance diseaseresistance in plants.

Proteins and Nucleic Acid Sequences According to the Invention

The NRC1 protein obtained from tomato shows low sequence identity (lessthan 25%) to NRG1 of tobacco. NRC1 also contains a larger number ofLeucine Rich Repeats (LRR) than NRG1. The protein structure of NRC1 isshown in FIG. 1 and SEQ ID NO: 2.

In one embodiment of the invention nucleic acid sequences and amino acidsequences of NRC1 proteins are provided (including orthologs), as wellas methods for isolating or identifying orthologs of NRC1 in other plantspecies, such as other Solanaceae, preferably potato. Equally, methodsfor isolating or identifying other NRC1 alleles, such as alleles fromother tomato species, varieties, lines or accessions are providedherein.

In one embodiment NRC1 proteins are provided. “NRC1 proteins” comprisethe protein depicted in SEQ ID NO: 2 (wild type) and 4 (constitutivemutant), as well as fragments and variants thereof. Variants of NRC1include, for example, proteins having at least 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 98, 99%, or more, amino acid sequenceidentity (over the entire length) to SEQ ID NO: 2 and/or 4. Amino acidsequence identity is determined by pairwise alignment using theNeedleman and Wunsch algorithm and GAP default parameters as definedabove. Variants of NRC1 can be obtained from various sources, such asexisting sequence databases, from other plant species (especially otherspecies of Solanaceae, such as potato) or other varieties or they can bemade by de novo synthesis, mutagenesis and the like. For example, SEQ IDNO: 4, a constitutively active NRC1 mutant, which is a variant of SEQ IDNO: 2, and was made by targeted mutagenesis using overlap PCR (seeExamples). The NRC1 proteins according to the invention may, thus, beisolated from natural sources, synthesized de novo by chemical synthesis(using e.g. a peptide synthesizer such as supplied by AppliedBiosystems) or produced by recombinant host cells by expressing thenucleic acid sequence encoding the NRC1 protein, fragment or variant.

NRC1 variants may comprise conservative amino acid substitutions withinthe categories basic (e. g. Arg, His, Lys), acidic (e. g. Asp,Glu),nonpolar (e. g. Ala, Val, Trp, Leu, Ile, Pro, Met, Phe, Trp) or polar(e. g. Gly, Ser, Thr, Tyr, Cys, Asn, Gln). In addition non-conservativeamino acid substitutions fall within the scope of the invention.

The functionality of any NRC1 protein, variant or fragment, can bedetermined using various methods. For example, transient or stableoverexpression in plant cells can be used to test whether the proteinhas activity in planta. Functionality is preferably tested in the sameplant species from which the protein is obtained. Thus, for exampletransient or stable expression can be used to determine whether an HRdevelops and/or whether resistance is enhanced, indicatingfunctionality. Alternatively, silencing of the endogenous genes or genefamily will show whether the NRC1 protein is functional. For example,VIGS can be used in a variety of Solanaceae, such as potato, tomato andtobacco (see Brigneti et al., 2004, Plant Journal 39: 264;Faivre-Rampant et al. Plant Physiology 134: 1308-1316; Baulcombe 1999,Curr. Opinion. Plant Biol. 2: 109-113; Lu et al. 2003, EMBO J.22:5690-5699), in model organisms such as Arabidopsis (Turnage et al.2002, Plant J. 30: 107-114), in monocots such as barley (Holzberg et al.2002, Plant J. 30: 315-327). Alternatively, silencing vectors comprisingsense and/or antisense fragments of an NRC1 gene can be used totransform plant cells (see below), followed by an assay to determinedwhether the capability to develop HR lesions and/or disease resistanceis modified.

In a preferred embodiment variants of NRC1 include NRC1 proteins whichare constitutively active in plant cells, such as the NRC1 proteinprovided in SEQ ID NO: 4, which comprises a single amino acidsubstitution in the MHD domain (D481V) (see FIG. 1). The constitutiveactivity can be tested by determining whether the protein is capable ofeliciting an HR in plant tissue, in the absence of elicitor. Forexample, Agroinfiltration of a 35S:NRC1 construct, as described in theExamples, can be used to infiltrate leaf tissue. Other constitutivelyactive NRC1 proteins can be made, by either random mutagenesis followedby activity testing (as described in Bendahame et al., 2002, p196) or bysite directed mutagenesis of single amino acids in the MHD domain (anyone of amino acids VHD or VHDM may be replaced with another amino acid),the NB-ARC domain, e.g. in the RNBS-D domain (amino acids FLYFGTFPRGY),or one of the 13 LRR domains (see FIG. 1). Alternatively, nucleic acidsequences encoding constitutively active NRC1 proteins can be obtainedfrom plants, for example by mutagenizing seeds and screening these forthe presence of a spontaneous lesion phenotype (for example microscopiclesions), see e.g. Sharino et al. (2002, The Plant Cell 14: 3149-3162)and further below.

In one embodiment also chimeric NRC1 proteins are provided. Suchproteins comprise at least a CC domain, a NB-ARC domain and preferablyat least 13 LRRs. A CC-, NB-ARC- and LRR-domain preferably refers toamino acid motifs comprising at least 30, 40, 50, 60, 70, 80, 90, 95,98, 99%, or more, amino acid sequence identity to amino acids 1-150, toamino acids 151-508, or to amino acids 509-846 of SEQ ID NO: 2respectively. Domains may thus be exchanged (domain swapping) betweenNRC1 proteins or between NRC1 proteins and other CC-NB-LRR or TIR-NB-LRRproteins, as long as the functionality of the resulting chimeric proteinis essentially similar to that of NRC1, or preferably to NRC1^(D481V).Most preferably, the chimeric protein retains the ability to confer orenhance disease resistance when it is produced by recombinant plantcells, as described below.

“Fragments” of NRC1 proteins and of variants of NRC1 proteins, asdescribed above, comprise fragments of 100, 150, 200, 300, 400, 500,600, 700, 800, 850, 855 contiguous amino acids or more. Preferably, suchfragments are functional in plant tissue, i.e. they are capable ofconferring or enhancing pathogen resistance when produced in plantcells. Fragments may also be used to make chimeric proteins, asdescribed above.

In another embodiment isolated nucleic acid sequences encoding any ofthe above proteins, variants or fragments are provided, such as cDNA,genomic DNA and RNA sequences. Due to the degeneracy of the genetic codevarious nucleic acid sequences may encode the same amino acid sequence.Any nucleic acid sequence encoding NRC1 proteins or variants are hereinreferred to as “NRC1”. The nucleic acid sequences provided includenaturally occurring, artificial or synthetic nucleic acid sequences.Examples of nucleic acid sequences encoding NRC1 proteins are providedfor in SEQ ID NO: 1 and 3. It is understood that when sequences aredepicted in as DNA sequences while RNA is referred to, the actual basesequence of the RNA molecule is identical with the difference thatthymine (T) is replace by uracil (U).

Also included are variants and fragments of NRC1 nucleic acid sequences,such as nucleic acid sequences hybridizing to NRC1 nucleic acidsequences under stringent hybridization conditions as defined. Variantsof NRC1 nucleic acid sequences also include nucleic acid sequences whichhave a sequence identity to SEQ ID NO: 1 or 3 (over the entire length)of at least 50% or more, preferably at least 55%, 60%, 70%, 80%, 90%,95%, 99%, 99.5%, 99.8% or more. In a preferred embodiment, variants ofNRC1 encode constitutively active NRC1 proteins as described. It isclear that many methods can be used to identify, synthesise or isolatevariants or fragments of NRC1 nucleic acid sequences, such as nucleicacid hybridization, PCR technology, in silico analysis and nucleic acidsynthesis, and the like.

The nucleic acid sequence, particularly DNA sequence, encoding the NRC1proteins of this invention can be inserted in expression vectors toproduce high amounts of NRC1 proteins (or e.g. chimeric NRC1 proteins),as described below. For optimal expression in a host the NRC1 DNAsequences can be codon-optimized by adapting the codon usage to thatmost preferred in plant genes, particularly to genes native to the plantgenus or species of interest (Bennetzen & Hall, 1982, J. Biol. Chem.257, 3026-3031; Itakura et al., 1977 Science 198, 1056-1063.) usingavailable codon usage tables (e. g. more adapted towards expression incotton, soybean corn or rice). Codon usage tables for various plantspecies are published for example by Ikemura (1993, In “Plant MolecularBiology Labfax”, Croy, ed., Bios Scientific Publishers Ltd.) andNakamura et al. (2000, Nucl. Acids Res. 28, 292.) and in the major DNAsequence databases (e.g. EMBL at Heidelberg, Germany). Accordingly,synthetic DNA sequences can be constructed so that the same orsubstantially the same proteins are produced. Several techniques formodifying the codon usage to that preferred by the host cells can befound in patent and scientific literature. The exact method of codonusage modification is not critical for this invention.

Small modifications to a DNA sequence such as described above can beroutinely made, i.e., by PCR-mediated mutagenesis (Ho et al., 1989, Gene77, 51-59., White et al., 1989, Trends In genet. 5, 185-189). Moreprofound modifications to a DNA sequence can be routinely done by denovo DNA synthesis of a desired coding region using availabletechniques.

Also, the NRC1 nucleic acid sequences can be modified so that theN-terminus of the NRC1 protein has an optimum translation initiationcontext, by adding or deleting one or more amino acids at the N-terminalend of the protein. Often it is preferred that the proteins of theinvention to be expressed in plants cells start with a Met-Asp orMet-Ala dipeptide for optimal translation initiation. An Asp or Alacodon may thus be inserted following the existing Met, or the secondcodon, Val, can be replaced by a codon for Asp (GAT or GAC) or Ala (GCT,GCC, GCA or GCG). The DNA sequences may also be modified to removeillegitimate splice sites.

“Fragments” of NRC1 nucleic acid sequences include fragments of at least10, 12, 15, 16, 18, 20, 30, 40, 50, 100, 200, 500, 1000, 1500, 2000 ormore consecutive nucleotides of SEQ ID NO: 1 or 3, or of variants of SEQID NO: 1 or 3. Short fragments can for example be used as PCR primers orhybridization probes.

In another embodiment of the invention PCR primers and/or probes andkits for detecting the NRC1 DNA or RNA sequences are provided.Degenerate or specific PCR primer pairs to amplify NRC1 DNA from samplescan be synthesized based on SEQ ID NO: 1 or 3 (or variants thereof) asknown in the art (see Dieffenbach and Dveksler (1995) PCR Primer: ALaboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson atal. (2000) PCR-Basics: From Background to Bench, First Edition, SpringerVerlag, Germany). For example, any stretch of 9, 10, 11, 12, 13, 14, 15,16, 18 or more contiguous nucleotides of SEQ ID NO: 1 or 3 (or thecomplement strand) may be used as primer or probe. Likewise, DNAfragments of SEQ ID NO: 1 or 3 (or variants thereof) can be used ashybridization probes. An NRC1 detection kit may comprise either NRC1specific primers and/or NRC1 specific probes, and an associated protocolto use the primers or probe to detect NRC1 DNA in a sample. Such adetection kit may, for example, be used to determine, whether a planthas been transformed with an NRC1 gene (or part thereof) of theinvention. Because of the degeneracy of the genetic code, some aminoacid codons can be replaced by others without changing the amino acidsequence of the protein.

In yet another embodiment a method for identifying and using orthologsor alleles of the tomato NRC1 gene (SEQ ID NO: 1 and 3) is provided. Themethod comprises the steps of:

-   -   a) obtaining or identifying a nucleic acid sequence comprising        at least 70% nucleic acid identity to SEQ ID NO: 1 and/or 3 (or        a higher percentage sequence identity, as indicated above),    -   b) optionally modifying the nucleic acid sequence to encode a        constitutively active NRC1 protein, and    -   c) using the nucleic acid sequence of a) to generate expression        and/or silencing vectors, or using the nucleic acid sequence        of b) to generate expression vectors,    -   d) using one or more vectors of c) to transform a plant or plant        cell(s), preferably of the plant species from which the nucleic        acid was obtained,    -   e) analysing the capability of the transformed plant/plant        tissue to develop HR lesions (i.e. the HR lesion phenotype,        which can optionally be quantified) and/or the disease        resistance of the transformants in order to determine or verify        the gene function in planta and/or to generate transgenic plants        having enhanced disease resistance;    -   f) optionally selecting those alleles or orthologs for further        use which confer enhanced disease resistance to the transgenic        plant but which, upon expression, confer a weak HR phenotype        (i.e. cause no or a reduced HR lesion phenotype).

Thus, NRC1 alleles or orthologs, which upon expression in plants resultin fewer and/or smaller HR lesions than seen upon expression of SEQ IDNO: 1 or 3, or upon expression of the wild type NRC1 allele obtainedfrom the host species to be transformed, can be identified using thismethod. Most preferably, NRC1 alleles or orthologs are identified whichcause no HR lesions, or at least no macroscopically visible HR lesions,upon expression, but which still confer enhanced disease resistance.

The HR phenotype of different NRC1 alleles and/or orthologs can becompared by making expression vectors using the same promoters,transforming a host plant with these, and by comparing the HR lesionphenotype between these transgenic plants. When different alleles arecompared in transgenic Solanaceae plants (e.g. under control of aconstitutive or inducible promoter), the HR lesion phenotype oftransformants expressing SEQ ID NO: 1 or 3 is preferably used asreference. Alternatively, the wild type allele obtained from the hostspecies to be transformed can be used as reference. Those alleles whichprovide fewer and/or smaller HR lesions compared to SEQ ID NO: 1 or 3,or fewer and/or smaller HR lesions than caused by expression of the wildtype allele obtained from the species transformed, can then be selectedfor further use. For example transgenic plants expressing these can bemade as described below.

Especially alleles from tomato and orthologs from potato may be obtainedor identified using e.g. NRC1-specific PCR primers or probes, orbioinformatics analysis in silico. Also genetic mapping may be used tomap the NRC1 locus in the plant (e.g. tomato or potato) genome, wherebysequences may be obtained by linking the genomic map to existing genomesequencing databases (e.g. developed in the tomato sequencing project).Such alleles and/or orthologs may be especially suitable for generatingplants with enhanced disease resistance.

When potato orthologs of NRC1 are identified in the above method, theseorthologs (or variants of these orthologs) are preferably used togenerate plants having enhanced resistance to Phytophthora infestans.

Chimeric Genes, Expression Vectors and Recombinant Organisms Accordingto the Invention

In one embodiment of the invention nucleic acid sequences encoding NRC1proteins (including variants or fragments), as described above, are usedto make chimeric genes, and vectors comprising these for transfer of thechimeric gene into a host cell and production of the NRC1 protein(s) inhost cells, such as cells, tissues, organs or organisms derived fromtransformed cell(s). Vectors for the production of NRC1 protein (orprotein fragments or variants) in plant cells are herein referred to asi.e. “expression vectors”. Host cells are preferably plant cells and,but microbial hosts (bacteria, e.g. Agrobacterium, yeast, fungi, etc.)are also envisaged.

Any plant may be a suitable host, such as monocotyledonous plants ordicotyledonous plants, but most preferably the host plant belongs to thefamily Solanaceae. For example, the plant belongs to the genus Solanum(including Lycopersicon), Nicotiana, Capsicum, Petunia and other genera.The following host species may suitably be used: Tobacco (Nicotianaspecies, e.g. N. benthamiana, N. plumbaginifolia, N. tabacum, etc.),vegetable species, such as tomato (L. esculentum, syn. Solanumlycopersicum) such as e.g. cherry tomato, var. cerasiforme or curranttomato, var. pimpinellifolium) or tree tomato (S. betaceum, syn.Cyphomandra betaceae), potato (Solanum tuberosum), eggplant (Solanummelongena), pepino (Solanum muricatum), cocona (Solanum sessiliflorum)and naranjilla (Solanum quitoense), peppers (Capsicum annuum,Capsicumfrutescens, Capsicum baccatum), ornamental species (e.g. Petuniahybrida, Petunia axillaries, P. integrifolia).

Alternatively, the plant may belong to any other family, such as to theCucurbitaceae or Gramineae. Suitable host plants include for examplemaize/corn (Zea species), wheat (Triticum species), barley (e.g. Hordeumvulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye(Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypiumspecies, e.g. G. hirsutum, G. barbadense), Brassica spp. (e.g. B. napus,B. juncea, B. oleracea, B. rapa, etc), sunflower (Helianthus annus),safflower, yam, cassava, alfalfa (Medicago sativa), rice (Oryza species,e.g. O. sativa indica cultivar-group or japonica cultivar-group), foragegrasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species(Pinus, poplar, fir, plantain, etc), tea, coffea, oil palm, coconut,vegetable species, such as pea, zucchini, beans (e.g. Phaseolusspecies), cucumber, artichoke, asparagus, broccoli, garlic, leek,lettuce, onion, radish, turnip, Brussels sprouts, carrot, cauliflower,chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearingplants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry,apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime,nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamentalspecies (e.g. Rose, Petunia, Chrysanthemum, Lily, Gerbera species),herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g. species ofPopulus, Salix, Quercus, Eucalyptus), fibre species e.g. flax (Linumusitatissimum) and hemp (Cannabis sativa), or model organisms, such asArabidopsis thaliana.

Preferred hosts are “crop plants”, i.e. plant species which iscultivated and bred by humans. A crop plant may be cultivated for foodpurposes (e.g. field crops), or for ornamental purposes (e.g. productionof flowers for cutting, grasses for lawns, etc.). A crop plant asdefined herein also includes plants from which non-food products areharvested, such as oil for fuel, plastic polymers, pharmaceuticalproducts, cork and the like.

The construction of chimeric genes and vectors for, preferably stable,introduction of NRC1 protein encoding nucleic acid sequences into thegenome of host cells is generally known in the art. To generate achimeric gene the nucleic acid sequence encoding a NRC1 protein (orvariant or fragment) is operably linked to a promoter sequence, suitablefor expression in the host cells, using standard molecular biologytechniques. The promoter sequence may already be present in a vector sothat the NRC1 nucleic sequence is simply inserted into the vectordownstream of the promoter sequence. The vector is then used totransform the host cells and the chimeric gene is inserted in thenuclear genome or into the plastid, mitochondrial or chloroplast genomeand expressed there using a suitable promoter (e.g., Mc Bride et al.,1995 Bio/Technology 13, 362; U.S. Pat. No. 5,693,507). In one embodimenta chimeric gene comprises a suitable promoter for expression in plantcells or microbial cells (e.g. bacteria), operably linked thereto anucleic acid sequence encoding a NRC1 protein according to theinvention, optionally followed by a 3′ nontranslated nucleic acidsequence.

The NRC1 nucleic acid sequence, preferably the NRC1 chimeric gene,encoding an functional NRC1 protein (or in certain embodiments aconstitutively active NRC1 protein), can be stably inserted in aconventional manner into the nuclear genome of a single plant cell, andthe so-transformed plant cell can be used in a conventional manner toproduce a transformed plant that has an altered phenotype due to thepresence of the NRC1 protein in certain cells at a certain time. In thisregard, a T-DNA vector, comprising a nucleic acid sequence encoding aNRC1 protein, in Agrobacterium tumefaciens can be used to transform theplant cell, and thereafter, a transformed plant can be regenerated fromthe transformed plant cell using the procedures described, for example,in EP 0 116 718, EP 0 270 822, PCT publication WO84/02913 and publishedEuropean Patent application EP 0 242 246 and in Gould et al. (1991,Plant Physiol. 95, 426-434). The construction of a T-DNA vector forAgrobacterium mediated plant transformation is well known in the art.The T-DNA vector may be either a binary vector as described in EP 0 120561 and EP 0 120 515 or a co-integrate vector which can integrate intothe Agrobacterium Ti-plasmid by homologous recombination, as describedin EP 0 116 718.

Preferred T-DNA vectors each contain a promoter operably linked to NRC1encoding nucleic acid sequence (e.g. encoding SEQ ID NO: 2 or SEQ ID NO:4) between T-DNA border sequences, or at least located to the left ofthe right border sequence. Border sequences are described in Gielen etal. (1984, EMBO J 3,835-845). Of course, other types of vectors can beused to transform the plant cell, using procedures such as direct genetransfer (as described, for example in EP 0 223 247), pollen mediatedtransformation (as described, for example in EP 0 270 356 andWO85/01856), protoplast transformation as, for example, described inU.S. Pat. No. 4,684,611, plant RNA virus-mediated transformation (asdescribed, for example in EP 0 067 553 and U.S. Pat. No. 4,407,956),liposome-mediated transformation (as described, for example in U.S. Pat.No. 4,536,475), and other methods. For tomato or tobacco transformationsee also An G. et al., 1986, Plant Physiol. 81: 301-305; Horsch R. B. etal., 1988, In: Plant Molecular Biology Manual A5, Dordrecht,Netherlands, Kluwer Academic Publishers. pp 1-9; Koornneef M. et al.,1986, In: Nevins D. J. and R. A. Jones, eds. Tomato Biotechnology, NewYork, N.Y., USA, Alan R. Liss, Inc. pp 169-178). For potatotransformation see e.g. Sherman and Bevan (1988, Plant Cell Rep. 7:13-16).

Likewise, selection and regeneration of transformed plants fromtransformed cells is well known in the art. Obviously, for differentspecies and even for different varieties or cultivars of a singlespecies, protocols are specifically adapted for regeneratingtransformants at high frequency.

Besides transformation of the nuclear genome, also transformation of theplastid genome, preferably chloroplast genome, is included in theinvention. One advantage of plastid genome transformation is that therisk of spread of the transgene(s) can be reduced. Plastid genometransformation can be carried out as known in the art, see e.g. SidorovV A et al. 1999, Plant J. 19: 209-216 or Lutz K A et al. 2004, Plant J.37(6):906-13.

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants containing thetransgene. Single copy transformants can be selected, using e.g.Southern Blot analysis or PCR based methods or the Invader® Technologyassay (Third Wave Technologies, Inc.). Transformed cells and plants caneasily be distinguished from non-transformed ones by the presence of thechimeric gene. The sequences of the plant DNA flanking the insertionsite of the transgene can also be sequenced, whereby an “Event specific”detection method can be developed, for routine use. See for exampleWO0141558, which describes elite event detection kits (such as PCRdetection kits) based for example on the integrated sequence and theflanking (genomic) sequence.

The NRC1 nucleic acid sequence is inserted in a plant cell genome sothat the inserted coding sequence is downstream (i.e. 3′) of, and underthe control of, a promoter which can direct the expression in the plantcell. This is preferably accomplished by inserting the chimeric gene inthe plant cell genome, particularly in the nuclear or plastid (e. g.chloroplast) genome.

As the constitutive production of the NRC1 protein may leads to theinduction of cell death (e.g. microscopic lesions and/or macroscopiclesions) and/or may lower yield (see e.g. Rizhsky and Mittler, Plant MolBiol, 2001 46: 313-23), it is in one embodiment preferred to use apromoter whose activity is inducible. Examples of inducible promotersare wound-inducible promoters, such as the MPI promoter described byCordera et al. (1994, The Plant Journal 6, 141), which is induced bywounding (such as caused by insect or physical wounding), or the COMPTIIpromoter (WO0056897) or the PR1 promoter described in U.S. Pat. No.6,031,151. Alternatively the promoter may be inducible by a chemical,such as dexamethasone as described by Aoyama and Chua (1997, PlantJournal 11: 605-612) and in U.S. Pat. No. 6,063,985 or by tetracycline(TOPFREE or TOP 10 promoter, see Gatz, 1997, Annu Rev Plant PhysiolPlant Mol Biol. 48: 89-108 and Love et al. 2000, Plant J. 21: 579-88).Other inducible promoters are for example inducible by a change intemperature, such as the heat shock promoter described in U.S. Pat. No.5,447,858, by anaerobic conditions (e.g. the maize ADH1S promoter), bylight (U.S. Pat. No. 6,455,760), by pathogens (e.g. the gst1 promoter ofEP759085 or the vst1 promoter of EP309862) or by senescence (SAG12 andSAG13, see U.S. Pat. No. 5,689,042). Obviously, there are a range ofother promoters available.

In one embodiment preferably, a pathogen inducible promoter is used, asthereby the NRC1 protein (or variant or fragment) will only be producedfollowing pathogen attack of the plant tissue. Especially, promoters ofgenes which are upregulated quickly after pathogen attack are desired.Pathogen inducible promoters include, for example, the hsr203J, str246Cand sgd24 promoters from tobacco, EAS4 promoter described by Yin et al.(1997, Plant Physiology 115(2):437-51), the tap1 or tap2 promoter (Mohanet al., 1993, Plant Mol Biol. 1993 22 :475-90), the gst1 promoter orvariants thereof (Martini et al. 1993, Mol. Gen. Gen. 236, 179-186;Hennin C., 1997, Afstudeerwerk, Faculteit Landbouwkundige en ToegepasteBiologische Wetenschappen, University of Gent, Belgium), the WRKYpromoters (Eulgem et al., EMBO J., 1999, 18(17):4689-99 and chimericpromoters described in WO0029592). Promoters inducible by a particularplant pathogen may also be identified using known methods, such ascDNA-AFLP®.

Preferably, the promoter is inducible by a number of pathogens, i.e. itis inducible by a broad range of pathogens of the host plant. For eachparticular host plant species, a different promoter may be mostsuitable. For example, when tomato is used as a host, the promoter ispreferably induced upon at least one, but preferably more than onetomato pathogen. Especially, a promoter which is inducible by one ormore fungal plant pathogens and/or bacterial plant pathogens (especiallyby one or more biotrophic and/or hemi-biotrophic plant pathogens) ispreferred.

Detailed descriptions of plant pathogens, the disease symptoms caused bythem and their life cycles can be found for each plant species. Forexample, tomato pathogens are described in “Compendium of TomatoDiseases”, Editors Jones, Jones, Stall and Zitter, ISBN 0-89054-120-5,APS Press (on the world wide web at shopapspress/org). Potato pathogensare described in “Compendium of Potato Disease”, 2^(nd) edition, EditorsStevenson, Franc and Weingartner, APS Press, ISBN 0-89054-275-9.

Pathogens of tomato include, for example, the following fungal andbacterial species and viruses (non-limiting): Botrytis cinerea(fungus/necrotroph); Colletotrichum coccodes (fungus/necrotroph);Alternaria alternata (fungus); Alternaria solani (fungus/necrotroph);Stemphylium solani; Phytophthora infestans (oomycte/hemibiotroph);Septoria lycopersici; Cladosporium fulvum, (fungus/hemibiotroph);Phytophthora parasitica; Oidium lycopersicum (biotroph); Fusariumoxysporum; Sclerotium rolfsii; Pythium; Rhizoctonia (fungus/necrotroph);Corynebacterium michiganense (bacterium); Pseudomonas syringae pv tomatoor pv syringae (bacterium/biotroph); Pseudomonas solanacearum;Pseudomonas corrugate; Clavibacter Xanthomonas campestris(bacterium/biotroph); Verticillium (fungus), tomato spotted wilt virus(TSWV); Tobacco or tomato mosaic viruses (TobMV, TomMV).

Pathogens of potato include, for example, various fungi, bacteria,nematodes and viruses, such as: Phytophthora infestans(oomycte/hemibiotroph), nematodes (biotrophic); Erwinia carotovora(bacterium); Colletotrichum coccodes (fungus); Rhizoctonia solani(fungus/necrotroph); Verticillium dahliae (fungus); Streptomycesscabies; Alternaria solani (fungus/necrotroph); Pythium; Spongosporasubterranean; PVX and PVY; Potato Leafroll Virus (PLRV); etc.

See also on the world wide web at apsnet.org/online/common/toc.asp forplant diseases of various plant species. Thus, in one embodiment thepromoter is preferably inducible by one or more of the above pathogens,most preferably at least by one or more of the above biotrophic and/orhemibiotrophic pathogens.

Alternatively, a host plant may comprise various NRC1 transgenes, eachunder control of a different pathogen inducible promoter, to ensure thatNRC1 protein is produced following attack by a variety of pathogens. Forexample, for transformation of tomato, one promoter may be inducible byPhytophthora and one by Cladosporium.

The word “inducible” does not necessarily require that the promoter iscompletely inactive in the absence of the inducer stimulus. A low levelnon-specific activity may be present, as long as this does not result insevere yield or quality penalty of the plants. Inducible, thus,preferably refers to an increase in activity of the promoter, resultingin an increase in transcription of the downstream NRC1 coding regionfollowing contact with the inducer.

The most preferred combination herein is the use of a pathogen induciblepromoter, operably linked to an NRC1 nucleic acid sequence which encodesa constitutively active NRC1 protein, as described above. In this caseupon pathogen attack the constitutively active NRC1 will be expressedresulting in a local HR (restricted to the site of pathogen attack)preventing further growth of any (hemi)-biotrophic pathogen.

In another embodiment constitutive promoters may be used, such as thestrong constitutive 35S promoters or enhanced 35S promoters (the “35Spromoters”) of the cauliflower mosaic virus (CaMV) of isolates CM 1841(Gardner et al., 1981, Nucleic Acids Research 9, 2871-2887), CabbB-S(Franck et al., 1980, Cell 21, 285-294) and CabbB-JI (Hull and Howell,1987, Virology 86, 482-493); the 35S promoter described by Odell et al.(1985, Nature 313, 810-812) or in U.S. Pat. No. 5,164,316, promotersfrom the ubiquitin family (e.g. the maize ubiquitin promoter ofChristensen et al., 1992, Plant Mol. Biol. 18, 675-689, EP 0 342 926,see also Comejo et al. 1993, Plant Mol. Biol. 23, 567-581), the gos2promoter (de Pater et al., 1992 Plant J. 2, 834-844), the emu promoter(Last et al., 1990, Theor. Appl. Genet. 81, 581-588), Arabidopsis actinpromoters such as the promoter described by An et al. (1996, Plant J.10, 107.), rice actin promoters such as the promoter described by Zhanget al.(1991, The Plant Cell 3, 1155-1165) and the promoter described inU.S. Pat. No. 5,641,876 or the rice actin 2 promoter as described inWO070067; promoters of the Cassava vein mosaic virus (WO 97/48819,Verdaguer et al. 1998, Plant Mol. Biol. 37, 1055-1067), the pPLEX seriesof promoters from Subterranean Clover Stunt Virus (WO 96/06932,particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g.,pAdh1S (GenBank accession numbers X04049, X00581), and the TR1′ promoterand the TR2′ promoter (the “TR1′ promoter” and “TR2′ promoter”,respectively) which drive the expression of the 1′ and 2′ genes,respectively, of the T-DNA (Velten et al., 1984, EMBO J 3, 2723-2730),the Figwort Mosaic Virus promoter described in U.S. Pat. No. 6,051,753and in EP426641, histone gene promoters, such as the Ph4a748 promoterfrom Arabidopsis (PMB 8: 179-191), or others. In a preferred embodimentthe AA6 promoters, as described in PCT/NL2005/050083 (filed 16 Dec.2005) are used.

Alternatively, a promoter can be utilized which is not constitutive butrather is specific for one or more tissues or organs of the plant(tissue preferred/tissue specific, including developmentally regulatedpromoters), for example leaf preferred, epidermis preferred, rootpreferred, flower tissue e.g. tapetum or anther preferred, seedpreferred, pod preferred, etc.), whereby the NRC1 gene is expressed onlyin cells of the specific tissue(s) or organ(s) and/or only during acertain developmental stage. For example, the NRC1 gene(s) can beselectively expressed in the leaves of a plant by placing the codingsequence under the control of a light-inducible promoter such as thepromoter of the ribulose-1, 5-bisphosphate carboxylase small subunitgene of the plant itself or of another plant, such as pea, as disclosedin U.S. Pat. No. 5,254,799 or Arabidopsis as disclosed in U.S. Pat. No.5,034,322.

In one embodiment the promoter of the endogenous NRC1 gene is used. Forexample, the promoter of the tomato NRC1 gene may be isolated andoperably linked to the coding region encoding NRC1 protein of SEQ ID NO:2 or 4. The NRC1 promoter (the upstream transcription regulatory regionof SEQ ID NO: 1 and 3) can be isolated from tomato plants using knownmethods, such as TAIL-PCR (Liu et al. 1995, Genomics 25(3):674-81; Liuet al. 2005, Methods Mol Biol. 286:341-8), Linker-PCR, or Inverse PCR(IPCR).

The NRC1 coding sequence is preferably inserted into the plant genome sothat the coding sequence is upstream (i.e. 5′) of suitable 3′ endnontranslated region (“3′end” or 3′UTR). Suitable 3′ends include thoseof the CaMV 35S gene (“3′ 35S”), the nopaline synthase gene (“3′ nos”)(Depicker et al., 1982 J. Molec. Appl. Genetics 1, 561-573.), theoctopine synthase gene (“3′ocs”) (Gielen et al., 1984, EMBO J. 3,835-845) and the T-DNA gene 7 (“3′ gene 7”) (Velten and Schell, 1985,Nucleic Acids Research 13, 6981-6998), which act as 3′-untranslated DNAsequences in transformed plant cells, and others. In one embodiment the3′UTR of the tomato NRC1 gene is used, as shown in SEQ ID NO: 3, fromnucleotide 2748 to nucleotide 3168, and as shown in SEQ ID NO: 5. TheNRC1 3′UTR is also an embodiment in itself herein, as it may also beused as 3′UTR in combination with other coding regions. Equally, anyvariant or fragment of SEQ ID NO: 5 is provided. A variant of SEQ ID NO:5 includes nucleic acid sequences comprising at least 40, 50, 60, 70,80, 90, 95, 98, 99% or more nucleic acid sequence identity to SEQ ID NO:5 (as determined using the Needleman and Wunsch algorithm and the GAPpenalties as defined above). Fragments include any nucleotide sequencescomprising at least 30, 50, 100, 150, 200, 300, 400 or more consecutivenucleotides of SEQ ID NO: 5, or of a variant of SEQ ID NO: 5.

Introduction of the T-DNA vector into Agrobacterium can be carried outusing known methods, such as electroporation or triparental mating.

A NRC1 encoding nucleic acid sequence can optionally be inserted in theplant genome as a hybrid gene sequence whereby the NRC1 sequence islinked in-frame to a (U.S. Pat. No. 5,254,799; Vaeck et al., 1987,Nature 328, 33-37) gene encoding a selectable or scorable marker, suchas for example the neo (or nptII) gene (EP 0 242 236) encoding kanamycinresistance, so that the plant expresses a fusion protein which is easilydetectable.

All or part of a NRC1 nucleic acid sequence, encoding a NRC1 protein (orvariant or fragment), can also be used to transform microorganisms, suchas bacteria (e.g. Escherichia coli, Pseudomonas, Agrobacterium,Bacillus, etc.), fungi, or algae or insects, or to make recombinantviruses. Transformation of bacteria, with all or part of a NRC1 nucleicacid sequence of this invention, incorporated in a suitable cloningvehicle, can be carried out in a conventional manner, preferably usingconventional electroporation techniques as described in Maillon et al.(1989, FEMS Microbiol. Letters 60, 205-210.) and WO 90/06999. Forexpression in prokaryotic host cell, the codon usage of the nucleic acidsequence may be optimized accordingly (as described for plants above).Intron sequences should be removed and other adaptations for optimalexpression may be made as known.

The DNA sequence of the NRC1 nucleic acid sequence can be furtherchanged in a translationally neutral manner, to modify possiblyinhibiting DNA sequences present in the gene part and/or by introducingchanges to the codon usage, e.g., adapting the codon usage to that mostpreferred by plants, preferably the specific relevant plant genus, asdescribed above.

In accordance with one embodiment of this invention, the NRC1 proteins(or chimeric proteins) are targeted to intracellular organelles such asplastids, preferably chloroplasts, mitochondria, or are secreted fromthe cell, potentially optimizing protein stability and/or expression.Similarly, the protein may be targeted to vacuoles. For this purpose, inone embodiment of this invention, the chimeric genes of the inventioncomprise a coding region encoding a signal or target peptide, linked tothe NRC1 protein coding region of the invention. Particularly preferredpeptides to be included in the proteins of this invention are thetransit peptides for chloroplast or other plastid targeting, especiallyduplicated transit peptide regions from plant genes whose gene productis targeted to the plastids, the optimized transit peptide of Capelladeset al. (U.S. Pat. No. 5,635,618), the transit peptide offerredoxin-NADP+oxidoreductase from spinach (Oelmuller et al., 1993,Mol. Gen. Genet. 237, 261-272), the transit peptide described in Wong etal. (1992, Plant Molec. Biol. 20, 81-93) and the targeting peptides inpublished PCT patent application WO 00/26371. Also preferred arepeptides signalling secretion of a protein linked to such peptideoutside the cell, such as the secretion signal of the potato proteinaseinhibitor II (Keil et al., 1986, Nucl. Acids Res. 14, 5641-5650), thesecretion signal of the alpha-amylase 3 gene of rice (Sutliff et al.,1991, Plant Molec. Biol. 16, 579-591) and the secretion signal oftobacco PR1 protein (Comelissen et al., 1986, EMBO J. 5, 37-40).Particularly useful signal peptides in accordance with the inventioninclude the chloroplast transit peptide (e.g. Van Den Broeck et al.,1985, Nature 313, 358), or the optimized chloroplast transit peptide ofU.S. Pat. No. 5,510,471 and U.S. Pat. No. 5,635,618 causing transport ofthe protein to the chloroplasts, a secretory signal peptide or a peptidetargeting the protein to other plastids, mitochondria, the ER, oranother organelle. Signal sequences for targeting to intracellularorganelles or for secretion outside the plant cell or to the cell wallare found in naturally targeted or secreted proteins, preferably thosedescribed by Klösgen et al. (1989, Mol. Gen. Genet. 217, 155-161),Klösgen and Weil (1991, Mol. Gen. Genet. 225, 297-304), Neuhaus & Rogers(1998, Plant Mol. Biol. 38, 127-144), Bih et al. (1999, J. Biol. Chem.274, 22884-22894), Morris et al. (1999, Biochem. Biophys. Res. Commun.255, 328-333), Hesse et al. (1989, EMBO J. 8, 2453-2461), Tavladoraki etal. (1998, FEBS Lett. 426, 62-66.), Terashima et al. (1999, Appl.Microbiol. Biotechnol. 52, 516-523), Park et al. (1997, J. Biol. Chem.272, 6876-6881), Shcherban et al. (1995, Proc. Natl. Acad. Sci USA 92,9245-9249).

To allow secretion of the NRC1 proteins to the outside of thetransformed host cell, an appropriate secretion signal peptide may befused to the amino terminal end (N-terminal end) of the NRC1 protein.Putative signal peptides can be detected using computer based analysis,using programs such as the program Signal Peptide search (SignalP V1.1or 2.0) (Von Heijne, Gunnar, 1986 and Nielsen et al., 1996).

In one embodiment, several NRC1 encoding nucleic acid sequences areco-expressed in a single host, optionally under control of differentpromoters. A co-expressing host plant is easily obtained by transforminga plant already expressing NRC1 protein of this invention, or bycrossing plants transformed with different NRC1 proteins of thisinvention. Alternatively, several NRC1 protein encoding nucleic acidsequences can be present on a single transformation vector or beco-transformed at the same time using separate vectors and selectingtransformants comprising both chimeric genes. Similarly, one or moreNRC1 encoding genes may be expressed in a single plant together withother chimeric genes, for example encoding other proteins which enhancedisease resistance or which are involved in the disease resistancesignalling pathway, or others.

It is understood that the different proteins can be expressed in thesame plant, or each can be expressed in a single plant and then combinedin the same plant by crossing the single plants with one another. Forexample, in hybrid seed production, each parent plant can express asingle protein. Upon crossing the parent plants to produce hybrids, bothproteins are combined in the hybrid plant.

Preferably, for selection purposes but also for weed control options,the transgenic plants of the invention are also transformed with a DNAencoding a protein conferring resistance to herbicide, such as abroad-spectrum herbicide, for example herbicides based on glufosinateammonium as active ingredient (e.g. Liberty® or BASTA; resistance isconferred by the PAT or bar gene; see EP 0 242 236 and EP 0 242 246) orglyphosate (e.g. RoundUp®; resistance is conferred by EPSPS genes, seee.g. EP 0 508 909 and EP 0 507 698). Using herbicide resistance genes(or other genes conferring a desired phenotype) as selectable markerfurther has the advantage that the introduction of antibiotic resistancegenes can be avoided.

Alternatively, other selectable marker genes may be used, such asantibiotic resistance genes. As it is generally not accepted to retainantibiotic resistance genes in the transformed host plants, these genescan be removed again following selection of the transformants. Differenttechnologies exist for removal of transgenes. One method to achieveremoval is by flanking the chimeric gene with lox sites and, followingselection, crossing the transformed plant with a CRErecombinase-expressing plant (see e.g. EP506763B1). Site specificrecombination results in excision of the marker gene. Another sitespecific recombination systems is the FLP/FRT system described inEP686191 and U.S. Pat. No. 5,527,695. Site specific recombinationsystems such as CRE/LOX and FLP/FRT may also be used for gene stackingpurposes. Further, one-component excision systems have been described,see e.g. WO9737012 or WO9500555).

Transformed Plant Cells/Plants/Seeds and Uses of the Nucleic AcidSequence and Proteins According to the Invention

In the following part the use of the NRC1 sequences according to theinvention to generate transgenic plant cells, plants, plant seeds, etc.and any derivatives/progeny thereof, with an enhanced diseasesresistance phenotype is described.

A transgenic plant with enhanced disease resistance can be generated bytransforming a plant host cell with a nucleic acid sequence encoding atleast one NRC1 protein under the control of a suitable promoter, asdescribed above, and regenerating a transgenic plant from said cell.

Preferred promoters are promoters which are inducible by external bioticand/or abiotic stimuli. Especially promoters which are pathogeninducible are preferred, as described above. Preferred promoter—NRC1combinations are:

-   -   a) a pathogen inducible promoter—nucleic acid sequence encoding        a constitutively active NRC1 protein;    -   b) a pathogen inducible promoter—nucleic acid sequence encoding        a wild type NRC1 protein;    -   c) the promoter of a plant NRC1 gene (preferably of the same        species which is to be transformed)—nucleic acid sequence        encoding a constitutively active NRC1 protein;    -   d) the promoter of a plant NRC1 gene (preferably of the same        species which is to be transformed)—nucleic acid sequence        encoding a wild type NRC1 protein;    -   e) a biotic stress inducible promoter (e.g. insect wounding        inducible, pathogen inducible, etc.)—nucleic acid sequence        encoding a constitutively active NRC1 protein;    -   f) a biotic stress inducible promoter (e.g. insect inducible,        pathogen inducible, etc.)—nucleic acid sequence encoding a wild        type NRC1 protein;    -   g) A constitutive promoter (e.g. 35S promoter)—nucleic acid        sequence encoding a wild type NRC1 protein;    -   h) A constitutive promoter (e.g. 35S promoter)—nucleic acid        sequence encoding an amino-acid sequence comprising at least 70%        amino acid sequence identity to SEQ ID NO:2 over the entire        length.    -   i) A pathogen-inducible promoter—nucleic acid sequence encoding        an amino-acid sequence comprising at least 70% amino acid        sequence identity to SEQ ID NO:2 over the entire length.    -   j) The promoter of a plant NRC1 gene—nucleic acid sequence        encoding an amino-acid sequence comprising at lest 70% amino        acid sequence identity to SEQ ID NO:2 over the entire length.

In one embodiment the transgenic plant may show either constitutive HRlesions or inducible HR lesions, and enhanced disease resistance to oneor more pathogens. However, it is also envisaged herein that no HRlesions or “weak” HR lesions (such as smaller lesions, e.g.micro-lesions, and/or a low lesion frequency) develop, while the plantstill shows enhanced disease resistance. NRC1 alleles or orthologswhich, upon expression in host plants under control of the identicalpromoters, result in fewer and/or smaller HR lesions than SEQ ID NO: 1or 3, or than the expression of the wild type NRC1 allele obtained fromthe same host species which is transformed, are particularly preferredherein, especially in approaches g) and h) above. Such alleles/orthologscan be referred to as NRC1 alleles conferring a “weak HR phenotype” in agiven host. Such NRC1 alleles or orthologs can be identified and/orisolated as described herein above. The HR phenotype of different NRC1alleles and/or orthologs can be compared by making expression vectorsusing these (preferably all nucleic acids which are to be compared areoperably linked to the identical promoters, e.g. 35S), transformingplants or plant tissue with these, and by comparing the HR lesionphenotype between these plants. For Solanaceae transformants, the HRlesion phenotype of transformants expressing SEQ ID NO: 1 or 3 ispreferably used as reference and any allele resulting in fewer and/orsmaller HR lesions upon expression under control of the same promoter isan allele conferring a weak HR phenotype. The HR lesion phenotype can becompared and optionally quantified using various methods, such asmicroscopy (optionally staining dead cells), visual scoring, countinglesions to calculate the number per cm², measuring the diameter of HRlesions, etc.

Preferably, the transgenic plants of the invention comprise enhanceddisease resistance against one or more pathogens, especially biotrophicand/or hemibiotrophic pathogens of the transgenic plant species. Thus,for example transgenic tomato or potato plants comprise enhancedresistance to at least one, or more, of the fungal, bacterial, nematodespecies and/or viral pathogens listed above, most preferably at leastagainst one or several biotrophic and/or hemibiotrophic species.

“Disease resistance” or “increased/enhanced disease resistance” is usedherein to refer to an enhanced ability of transformants (compared towild type or control transformants) to withstand the attack of one ormore plant pathogens, or in other words, it refers to a significantreduction in disease symptoms in transformants compared tonon-transformed (or empty-vector transformed) controls. Diseaseresistance or enhanced disease resistance may be determined using avariety of methods. Often disease symptoms are scored visually (eitherin bioassays or in the field) by assessing the disease symptoms at oneor more time points after inoculation or contact with a pathogen.Alternative methods include methods whereby the pathogen is detected andoptionally quantified. A transgenic plant may thus show enhanced diseaseresistance if the amount of pathogen detected in/on the tissue issignificantly less compared to controls, or if the pathogen spread issignificantly slower than in controls. Ultimately, a significantincrease in average yield of transformants (e.g. at least 1%, 2%, 5%,10% or more) compared to controls, when grown under equivalent diseasepressure (preferably in the field) provides an indirect measurement ofenhanced disease resistance.

Thus, a plurality of transformed plants expressing NRC1 protein (or aconstitutively active NRC1 protein) show enhanced disease resistance ifthey show a significant reduction of disease symptoms, compared to theuntransformed or empty-vector transformed controls. Obviously,statistical analysis is required to determine whether significantdifference exist. Preferably, one or more disease symptoms are onaverage at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, or even 100%lower in NRC1 transformants than in the control plants. As the diseaseassay is different for every host-pathogen combination, no specificprotocol can be provided, but the skilled person knows how to determinewhether transformants show significantly enhanced disease resistance toone or more pathogens. Bioassays as known in the art for eachplant-pathogen combination can be used to compare resistance oftransgenic plants to suitable controls.

As the NRC1 protein may in some embodiments result in HR lesions in theabsence of pathogen (for example if the NRC1 gene is under the controlof a constitutive promoter): it may in certain embodiments be importantto differentiate between symptoms caused by NRC1 expression and symptomscaused by pathogen infection and spread. It may, therefore, be preferredto use methods which detect the pathogen itself (rather than necrosis onthe plant tissue) and to compare the amount of pathogen present or thespeed of pathogen spread. For example, bioassays may be used wherein thepathogen can be detected by staining. In the examples a transgenic C.fulvum race is used which expresses GUS. Fungal mycelium can, therefore,be visualized using X-gluc staining of the inoculated plant tissue. Asignificant reduction of fungal mycelium in the transgenic plantscompared to the controls indicates an enhanced resistance to the fungus.

It is also an embodiment to generate transgenic plants which expressseveral NRC1 proteins, preferably under the control of differentpromoters, such as different pathogen inducible promoters.

The disease resistance phenotype can be fine-tuned by expressing asuitable amount of NRC1 protein at a suitable time and location. Suchfine-tuning may be done by determining the most appropriate promoter fora particular host-pathogen combination and also by selecting transgenic“events” which show the desired expression level. A too low level ofNRC1 protein or too slow induction of NRC1 protein production followingpathogen attack may be insufficient to enhance disease resistancelevels. On the other hand, a too high protein level or expression attimes and locations devoid of pathogen attack, may result inagronomically undesired phenotypes, such as lesions in leaves or fruitin the absence of pathogens and yield penalties. However, the skilledperson can easily generate plants having enhanced disease resistance,but which at the same time are agronomical acceptable. Optimal NRC1alleles may be isolated or identified as described, e.g. allelesproviding high resistance levels and only a weak HR phenotype.

Transformants expressing desired levels of the NRC1 protein are selectedby e.g. analysing copy number (Southern blot analysis), mRNA transcriptlevels (e.g. RT-PCR using NRC1 primer pairs or flanking primers) or byanalysing the presence and level of NRC1 protein in various tissues(e.g. SDS-PAGE; ELISA assays, etc). For regulatory reasons, preferablysingle copy transformants are preferably selected and the sequencesflanking the site of insertion of the chimeric gene is analysed,preferably sequenced to characterize the “event”. High or moderate NRC1expressing transgenic events are selected for furthercrossing/backcrossing/selfing until a high performing elite event with astable NRC1 transgene is obtained.

Transformants expressing one or more NRC1 genes according to theinvention may also comprise other transgenes, such as other genesconferring disease resistance or conferring tolerance to other bioticand/or abiotic stresses. To obtain such plants with “stacked”transgenes, other transgenes may either be introgressed into the NRC1transformants, or the NRC1 transformants may be transformed subsequentlywith one or more other genes, or alternatively several chimeric genesmay be used to transform a plant line or variety. For example, severalchimeric genes may be present on a single vector, or may be present ondifferent vectors which are co-transformed.

In one embodiment the following genes are combined with one or more NRC1genes according to the invention: known disease resistance genes,especially genes conferring enhanced resistance to necrotophicpathogens, virus resistance genes, insect resistance genes, abioticstress resistance genes (e.g. drought tolerance, salt tolerance, heat-or cold tolerance, etc.), herbicide resistance genes, and the like. Thestacked transformants may thus have an even broader biotic and/orabiotic stress tolerance, to pathogen resistance, insect resistance,nematode resistance, salinity, cold stress, heat stress, water stress,etc. Also, NRC1 silencing approaches may be combined with NRC1expression approaches in a single plant. For example, NRC1overexpression in roots or tubers may confer or enhance root or tuberresistance to soil pathogens. At the same time downregulation of NRC1 inaerial parts may confer or enhance resistance to necrotrophic pathogens(or vice versa).

It is also possible to introduce or introgress the NRC1 gene into aplant breeding line which already has a certain level of diseaseresistance. For durability of disease resistance in the field, it may bedesirable to stack several disease resistance mechanisms in a plant,preferably whereby the resistance sources have different underlyingmolecular mechanisms.

Whole plants, seeds, cells, tissues and progeny (such as F1 hybrids, F2seeds/plants, etc.) of any of the transformed plants described above areencompassed herein and can be identified by the presence of thetransgene in the DNA, for example by PCR analysis using total genomicDNA as template and using NRC1 specific PCR primer pairs. Also “eventspecific” PCR diagnostic methods can be developed, where the PCR primersare based on the plant DNA flanking the inserted chimeric gene, see U.S.Pat. No. 6,563,026. Similarly, event specific AFLP fingerprints or RFLPfingerprints may be developed which identify the transgenic plant or anyplant, seed, tissue or cells derived there from.

It is understood that the transgenic plants according to the inventionpreferably do not show non-desired phenotypes, such as yield reduction,enhanced susceptibility to diseases (especially to necrotrophs) orundesired architectural changes (dwarfing, deformations) etc. and that,if such phenotypes are seen in the primary transformants, these can beremoved by normal breeding and selection methods(crossing/backcrossing/selfing, etc.). Any of the transgenic plantsdescribed herein may be homozygous or hemizygous for the transgene.

NRC1 Gene Silencing Approaches and Gene Silencing Vectors

It is a further embodiment of the invention to provide plants withenhanced disease resistance, especially against necrotrophic pathogens,whereby the plant is transformed with an NRC1 gene silencing vector.Without limiting the scope of the invention, it is thought thatsilencing of endogenous NRC1 genes or gene families results in theinability of the transgenic plant to trigger and/or mount an HRresponse. As necrotrophic pathogens require cell death for their growthand development, such plants may comprise enhanced resistance to one ormore necrotrophic pathogens.

“Gene silencing” refers to the down-regulation or complete inhibition ofgene expression of one or more target genes (e.g. endogenous NRC1genes). The use of inhibitory RNA to reduce or abolish gene expressionis well established in the art and is the subject of several reviews(e.g Baulcombe 1996, Stam et al. 1997, Depicker and Van Montagu, 1997).There are a number of technologies available to achieve gene silencingin plants, such as chimeric genes which produce antisense RNA of all orpart of the target gene (see e.g. EP 0140308 B1, EP 0240208 B1 and EP0223399 B1), or which produce sense RNA (also referred to asco-suppression), see EP 0465572 B1.

The most successful approach so far has however been the production ofboth sense and antisense RNA of the target gene (“inverted repeats”),which forms double stranded RNA (dsRNA) in the cell and silences thetarget gene. Methods and vectors for dsRNA production and gene silencinghave been described in EP 1068311, EP 983370 A1, EP 1042462 A1, EP1071762 A1 and EP 1080208 A1. A vector according to the invention may,therefore, comprise a transcription regulatory region which is active inplant cells operably linked to a sense and/or antisense DNA fragment ofa NRC1 gene according to the invention. Generally short (sense andantisense) stretches of the target gene sequence, such as 17, 18, 19,20, 21, 22 or 23 nucleotides of coding or non-coding sequence aresufficient. Longer sequences can also be used, such as 50, 100, 200 or250 nucleotides or more. Preferably, the short sense and antisensefragments are separated by a spacer sequence, such as an intron, whichforms a loop (or hairpin) upon dsRNA formation. Any short stretch of SEQID NO: 1 or 3, or variants thereof, may be used to make a NRC1 genesilencing vector and a transgenic plant in which one or more NRC1 genesare silenced in all or some tissues or organs (depending on thepromoters used). A convenient way of generating hairpin constructs is touse generic vectors such as pHANNIBAL and pHELLSGATE, vectors based onthe Gateway® technology (see Wesley et al. 2004, Methods Mol Biol.265:117-30; Wesley et al. 2003, Methods Mol Biol. 236:273-86 andHelliwell & Waterhouse 2003, Methods 30(4):289-95.), all incorporatedherein by reference.

By choosing conserved nucleic acid parts of the NRC1 gene, NRC1 familymembers in a host plant or plant parts can be silenced. Encompassedherein are also transgenic plants comprising a transcription regulatoryelement operably linked to a sense and/or antisense DNA fragment of aNRC1 gene and exhibiting enhanced resistance to one or more pathogens,especially necrotrophic pathogens.

Also, plants having enhanced resistance to one or more biotrophic and/orhemi-biotrophic pathogens and to one or more necrotrophic pathogens areprovided. Such plants can be generated by choosing appropriatepromoter—NRC1 gene combinations. For example a functional NRC1 proteinmay be produced in a certain tissue at a certain time (e.g. uponinduction or in aerial plant parts), providing resistance to biotrophicand/or hemibiotrophic pathogens, while the endogenous NRC1 gene(s) aresilenced in a different tissue and/or at a different time (e.g. inseedlings, in roots or tubers, etc.), thereby providing resistance toone or more necrotrophic pathogens. A single plant may, therefore,comprise both a chimeric NRC1 expressing transgene and an NRC1 silencinggene.

Mutant Alleles and Plants According to the Invention

It is also an embodiment of the invention to use non-transgenic methods,e.g. mutagenesis systems such as TILLING (Targeting Induced LocalLesions IN Genomics; McCallum et al., 2000, Nat Biotech 18:455, andMcCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporatedherein by reference) and selection to generate plant lines which producehigher levels of one or more NRC1 proteins according to the inventionand/or which produce a constitutively active NRC1 protein as described.Without limiting the scope of the invention, it is believed that suchplants could comprise point/deletion mutations in the promoter that arebinding sites for repressor proteins that would make the host NRC1 geneconstitutive or higher in expression. Constitutively active NRC1 mutantswill comprise mutations in the coding region, such as the MHD region.Preferably NRC1 protein levels in the mutant or parts of the mutant areat least about 2, 5, 10, 15%, or more, increased in the mutant comparedto non-mutant plants. TILLING uses traditional chemical mutagenesis(e.g. EMS mutagenesis) followed by high-throughput screening formutations (e.g. using Cel 1 cleavage of mutant-wild type DNAheteroduplexes and detection using a sequencing gel system), see e.g.Henikoff et al. Plant Physiology Preview May 21, 2004. Thus,non-transgenic plants, seeds and tissues comprising an enhanced NRC1gene expression in one or more tissues and comprising one or more of theNRC1 phenotypes according to the invention (enhanced disease resistanceand/or HR lesions) and methods for generating and identifying suchplants is encompassed herein.

The method comprises in one embodiment the steps of mutagenizing plantseeds (e.g. EMS mutagenesis), pooling of plant individuals or DNA, PCRamplification of a region of interest, heteroduplex formation andhigh-throughput detection, identification of the mutant plant,sequencing of the mutant PCR product. It is understood that othermutagenesis and selection methods may equally be used to generate suchmutant plants. Seeds may for example be radiated or chemically treatedand the plants screened for a modified phenotype, such as enhanceddisease resistance and/or HR lesions.

In another embodiment of the invention, the plant materials are naturalpopulations of the species or related species that comprisepolymorphisms or variations in DNA sequence at the NRC1 orthologouscoding and/or regulatory sequence. Mutations at the NRC1 gene target canbe screened for using a ECOTILLING approach (Henikoff et al 2004,supra). In this method natural polymorphisms in breeding lines orrelated species are screened for by the above described TILLINGmethodology, in which individual or pools of plants are used for PCRamplification of the NRC1 target, heteroduplex formation andhigh-throughput analysis. This can be followed up by selecting ofindividual plants having the required mutation that can be usedsubsequently in a breeding program to incorporate the desiredNRC1-orthologous allele to develop the cultivar with desired trait.

Mutant plants can be distinguished from non-mutants by molecularmethods, such as the mutation(s) present in the DNA, NRC1 proteinlevels, NRC1 RNA levels etc, and by the modified phenotypiccharacteristics.

The non-transgenic mutants may be homozygous or heterozygous for themutation.

Sequences Referred to

-   SEQ ID NO 1: coding region of the tomato NRC1 gene-   SEQ ID NO 2: amino acid sequence of the tomato NRC1 protein-   SEQ ID NO 3: full length cDNA of the tomato NRC1 gene (including 5′    and 3′ UTR)-   SEQ ID NO 4: amino acid sequence of the tomato NRC1^(D481V) protein-   SEQ ID NO 5: 3′UTR of the tomato NRC1 gene

The following non-limiting Examples illustrate the different embodimentsof the invention. Unless stated otherwise in the Examples, allrecombinant DNA techniques are carried out according to standardprotocols as described in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology,Current Protocols, USA. Standard materials and methods for plantmolecular work are described in Plant Molecular Biology Labfax (1993) byR. D. D. Croy, jointly published by BIOS Scientific Publications Ltd(UK) and Blackwell Scientific Publications, UK.

EXAMPLES 1. Material and Methods

1.1 VIGS in N. benthamiana, Agroinfiltration, HR and Disease Assays

Four-week-old N. benthamiana plants were agroinfiltrated with a 1:1mixture of pTV00-derived constructs (binary TRV RNA2 vector) andpBintra6 (binary TRV RNA1 vector) (Ratcliff et al., 2001 Plant J. 25,237-245), or a 1:1 mixture of pTRV-RNA2-derived constructs and pTRV-RNA1(Liu et al., 2002, Plant J. 31, 777-786; Liu et al., 2002, Plant J. 30,415-429). The following TRV constructs were used: TRV:NRC1, TRV:Cf-4 andTRV:SGT1 (Peart et al., 2002, Proc. Natl. Acad. Sci. USA 99,10865-10869), all in the TRV vector described by Ratcliffet al. (2001,supra) and TRV:EDS1, TRV:MEK2, TRV:RAR1 and TRV:NDR1 (Ekengren et al.,2003, Plant J. 36, 905-917), all in the TRV vector described by Liu etal. (2002, Plant J. 30, 415-429). For each TRV construct, in eachexperiment four plants were used. AvrPto and CP were agroinfiltrated inTRV-infected N. benthamiana expressing the resistance gene Pto (N.benthamiana:Pto (line 38-12 (Rommens et al., 1995, Plant Cell 7,1537-1544)) (Pedley and Martin, 2003, Annu. Rev. Phytopathol. 41,215-243) and Rx (N. benthamiana:Rx (line Rx-18) (Bendahtnane et al.,1999, Plant Cell 11, 781-791), respectively. In all other casesAgroinfiltration was performed in N. benthamiana expressing theresistance gene Cf-4 (N. benthamiana:Cf-4). Three weeks post TRVinoculation the third, fourth and fifth leaf above the inoculated leaveswere challenged with Agrobacterium tumefaciens that directs expressionof AvrPto (OD₆₀₀=0.06) (Tang et al., 1996, Science 274, 2060-2063), CP(OD₆₀₀=0.12) (Bendahmane et al., 1999, supra), Avr4 (OD₆₀₀=0.03), Cf-9and Avr9 (mixed in a 1:1 ratio, OD₆₀₀=0.2) (Van der Hoom et al., 2000Mol. Plant-Microbe Interact. 13, 439-446), LeEix2 and tvEix (mixed in a1:1 ratio, OD₆₀₀=1) (Ron and Avni, 2004, Plant Cell 16, 1604-1615), theβ-glucuronidase (GUS) gene (OD₆₀₀=2) (Van der Hoom et al., 2000, Mol.Plant-Microbe Interact. 13, 439-446), NRC1 and p19 (mixed in a 1:1ratio, OD₆₀₀=1) (Voinnet et al., 2003, Plant J. 33, 949-956), theconstitutively active NRC1^(481V) or the inactive NRC1^(K191R/D481V)double mutant (OD₆₀₀=2), LeMAPKKKα^(KD), LeMAPKKKα^(KD−) (Del Pozo etal., 2004, EMBO J. 23, 3072-3082) (both at OD₆₀₀=0.12) or LeMEK2DD andLeMEK2 (Del Pozo et al., 2004, supra) (both at OD₆₀₀=0.25). Two dayspost infiltration of LeMAPKKKα^(KD), LeMAPKKKα^(KD−), LeMEK2DD or LeMEK2the leaves were sprayed with a 7.5 μM solution of 17-β-estradiol inwater, containing silwet (4 μl/100 ml) (Del Pozo et al., 2004, supra).For protein injections, Avr4-HIS-FLAG-tagged protein was treated withenterokinase EK-max according to the manufacturer's recommendations(Invitrogen, Breda, NL) and 5 μM Avr4 protein in water, supplementedwith 0.2% tween (v/v) was used for injections. Three to five days postagroinfiltration or protein injection the leaves were examined for thedevelopment of an HR, or assayed for β-glucuronidase (GUS) activity.

1.2 VIGS in Tomato, HR and Disease Assays

For VIGS in tomato the pTRV-RNA1 and pTRV-RNA2 vectors described by Liuet al. (2002, Plant J. 30, 415-429) were used. The Cf-4 and NRC1fragments were excised from pTV00 by digestion with BamH1/Asp718 andinserted into BamH1/Asp718-digested pTRV-RNA2 (pYL156) (Liu et al.,2002, Plant J. 31, 777-786). To construct TRV:222-UTR, part of the3′-UTR of NRC1 was amplified using primers 222-3′UTR-F(5′-GTGGATCCGCAGGTTCAACCAGCCTGGT-3′; BamH1 site underlined) and222-3′UTR-R (5′-GTGGTACCCAAGTGACTTGTTCTGCTGT-3′; Asp718 site underlined)and to construct TRV:222-LRR, part of the NRC1 region coding the LRRswas amplified using primers 222-LRR-F(5′-GTGGATCCGTTAAGAGGCTGCAATTTCT-3′; BamH1 site underlined) and222-LRR-R (5′-GTGGTACCGATCTTCTCAAGTTTATCAC-3′; Asp718 site underlined).The PCR fragments were BamH1/Asp718-digested and inserted intoBamH1/Asp718-digested pTRV-RNA2. TRV:Prf construction has been described(Ekengren et al., 2003, Plant J. 36, 905-917). All plasmids weretransformed to A. tumefaciens strain GV3101 by electroporation (Takkenet al., 2000, Plant J. 24, 275-283). To establish VIGS in tomato,cotyledons of ten- to twelve-day-old tomato seedlings wereagroinfiltrated with a mixture of pTRV-RNA1 and the pTRV-RNA2-derivedconstructs (combined in a 1:1 ratio) (Liu et al., 2002, supra). For eachTRV construct either four Cf-4-containing tomato plants (Cf0 plantstransformed with Hcr9-4D (Cf-4)) (Thomas et al., 1997, Plant Cell 9,2209-2224), resistant to C. fulvum expressing Avr4, or fourCf-9-containing tomato plants (Cf0 plants transformed with Hcr9-9C(Cf-9)) (Jones et al., 1994, Science 266, 789-793), resistant to C.fulvum expressing Avr9, were used. As control Cf0 tomato plants(MM-Cf0), fully susceptible to C. fulvum, either TRV:00- orTRV:NRC1-inoculated were used. For disease assays, three weeks post TRVinoculation Cf0 and Cf-4-containing plants were inoculated with C.fulvum (De Wit, 1977, Neth. J. Plant Path. 83, 109-122). A C. fulvumrace 5-pGPD::GUS was used (expressing Avr4 and the β-glucuronidase geneunder control of the constitutive GPD promoter). Colonization of theleaflets was assessed two weeks later by X-gluc staining. In parallel,leaflets of the second, third or fourth compound leaf of the plants wereused for RT-PCR analysis to test for ‘knock down’ of the gene ofinterest (see below). For HR assays, leaflets of the third compound leafof TRV-infected Cf-4- or Cf-9-containing plants were injected with Avr4or Avr9, respectively. Both elicitors were injected into leaflets with amicro-syringe (Ito Corporation, Fuji, Japan). Avr4 was injected at aconcentration of 10 μM. at ten sites per leaflet and four leaflets perplant. For Avr9, eight times diluted apoplastic fluid containing about10 μM of Avr9, isolated from a compatible interaction between race 5 ofC. fulvum and Cf0 plants, was injected at eight sites per leaflet andfour leaflets per plant. Resistance against Pseudomonas syringae pv.tomato was assayed in tomato RG-PtoR (Pto/Pto, Prf/Prj), inoculated withTRV:00, TRV:Prf or TRV:NRC1. The inoculation procedure and thedetermination of bacterial colonization of the leaves were performed asdescribed previously (Ekengren et al., 2003, supra).

1.3 Binary 35S:NRC1 Vector Construction and Mutagenesis

Full length NRC1 cDNA was PCR-amplified using primers 222-Start-F(5′-GGGATCCATGGTTGATGTAGGGGTTGA-3′) and 222-Stop-R(5′-GTCACTGCAGACCTTTCTAAGAAGCTGTCTG-3′), thereby introducing NcoI andPstI restriction sites, respectively (restriction sites underlined). ThePCR fragment was NcoI/PstI-digested and inserted into NcoI/PstI-digestedpRH80 (Van der Hoom et al., 2000, Mol. Plant-Microbe Interact. 13,439-446). Subsequently, the construct was XbaI/KpnI-digested and theresulting fragment containing the 35S promoter, the NRC1 open readingframe and the NOS terminator (tNOS), was cloned into theXbaI/KpnI-digested pMOG800 binary vector (Honée et al., 1998, PlantPhysiol. 117, 809-820) to create plasmid NRC1(wt). To createconstitutively active binary NRC1^(D481V), the D481V mutation wasintroduced by overlap extension PCR (Higuchi et al., 1988, Nucleic AcidsRes. 16, 7351-7367) using the NRC1wt plasmid as a template and flankingprimers 222-Start-F and 222-Stop-R and mismatch primers 222MHD-F(5′-CAAAACTTGTCGTGTTCATGTCATGTTGTATGAG-3′) and 222MHD-R(5′-CCAGCAAAACTCATACAACATGACATGAACACGAC-3′) (mutation underlined).

The fragment was NcoI/PstI-digested, inserted into pRH80 and the35S-NRC1^(D481V)-tNOS fragment was excised and subsequently insertedinto pMOG800 as described above. In a similar way the P-loop mutantNRC1^(K191R), and the inactive double mutant NRC1^(K191R/D481V) werecreated. Here, the K191R mutation was introduced using mismatch primers222Ploop-F (5′-GGAATGCCTGGTCTTGGCAGAACTACACTAGC-3′) and 222Ploop-R(5′-GCTAGTGTAGTTCTGCCAAGACCAGGCATTCC-3′) (mutation underlined) withrespectively plasmid NRC1(wt) and NRC1^(D481V) as a template. Allconstructs were sequence-verified and transformed to A. tumefaciensstrain GV3101.

1.4 DNA Gel-Blot Analysis

Genomic DNA from N. benthamiana was isolated using the QIA-Gen DNAextraction protocol (Qiagen, Venlo, NL), whereas for tomato the standardprotocol described by (Sambrook and Russell, 2001, Molecular cloning: ALaboratory Manual, 3rd ed. (Cold Spring Harbor, N.Y., U.S.A.: ColdSpring Harbor Laboratory Press) was used. The DNA was digested withBamHI, HindIII, EcoRI, EcoRV or XbaI. The N. benthamiana gel-blot washybridized with the ³²P-labeled (Prime-a-gene Labeling System, Promega,Madison, Wis.) 252 bp fragment present in the TRV:NRC1 vector and theDNA gel-blot of tomato was hybridized with a ³²P-labeled probe of 1293bases corresponding to nucleotides 1876 to 3168 of the full length NRC1cDNA. Sites for the restriction enzymes used are not present in theprobes. Low stringency refers to washing at 55° C. in 2×SSC and 0.5%SDS. High stringency conditions consist of washing at 65° C. in 0.5×SSCand 0.5% SDS.

1.5 RT-PCRs to Show Silencing of NRC1 in Tomato

Four leaf discs (approximately 100 mg of tissue in total) were collectedfrom the second, third or fourth compound leaf of TRV-infected plants.Total RNA was extracted using the QIA-Gen RNAeasy extraction protocol(Qiagen, Venlo, NL) and treated with RNase-Free DNase (Bio-Rad,Veenendaal, NL). First strand cDNA was synthesized from 1 μg of totalRNA using the Bio-Rad cDNA synthesis kit (Bio-Rad, Veenendaal, NL) andRT-PCR was performed using the following cycles: 95° C. for 15 sec, 60°C. for 45 sec and 72° C. for 60 sec. The primers that were used (222F:5′-TGAGGTATATTGCTTTCTCATCTGAC-3′ and 222R:5′-AGCTATTTTCCCACGGATGCCCAG-3′) do not cover the fragment which isinserted in TRV:NRC1. Actin primers (ActinFnr182:5′-TATGGAAACATTGTGCTCAGTGG-3′ and ActinRnr183:5′-CCAGATTCGTCATACTCTGCC-3′) were used to check for the presence ofequal amounts of cDNA in the PCR reactions.

Example 2 Results

2.1 Tomato NRC1; a CC-NB-LRR Protein

cDNA-AFLP analysis was performed, followed by VIGS of the identifiedfragments of tomato in N. benthamiana:Cf-4. 20 cDNA fragments wereidentified of which VIGS affects the Cf-4/Avr4-induced HR. For one ofthese, NRC1, the full length cDNA was isolated, as depicted in SEQ IDNO: 3. The open reading frame is shown in SEQ ID NO: 1, which encodesthe NRC1 protein depicted in SEQ ID NO: 2.

The predicted primary structure of the NRC1 protein (SEQ ID NO: 2)typically resembles that of CC-NB-LRR resistance proteins (FIG. 1). NRC1has an amino-terminal coiled-coil (CC) domain, an NB-ARC (NucleotideBinding adapter shared by Apaf-1, R proteins and CED4) domain (Van derBiezen and Jones, 1998, Curr. Biol. 8, R226-R227; Aravind et al., 1999,Trends Biochem. Sci. 24, 47-53) and 13 imperfect leucine-rich repeats(LRRs). As indicated in FIG. 1, comparison with homologous NB-ARCdomains revealed the presence of a Kinase1A or P-loop motif, four RNBS(Resistance Nucleotide Binding Site) motifs and a GLPL and MHD motif(Meyers et al., 1999, Plant J. 20, 317-332; Meyers et al., 2003, PlantCell 15, 809-834).

The 252 bp cDNA-AFLP fragment present in the TRV:NRC1 vector used forVIGS codes for amino acids 599-681, which are located in LRRs four toseven.

Low stringency DNA gel-blot analysis of genomic DNA of tomato digestedwith the BamHI-, HindIII-, EcoRI-, EcoRV- and XbaI, was hybridized witha 1293 bp NRC1 cDNA fragment (nucleotides 1876 to 3168 of SEQ ID NO: 3)covering the NRC1 sequence present in the TRV:NRC1-, TRV:NRC1-LRR- andTRV:NRC1-UTR constructs (see below) as a probe. This Southern blotrevealed only one prominent band after a high stringency wash, whichindicates that NRC1 is a single-copy gene in tomato.

A gel blot of BamHI-, HindIII-, EcoRI-, EcoRV- and XbaI-digested genomicDNA of N. benthamiana was probed with the tomato NRC1 cDNA-AFLP fragmentpresent in the TRV vector and two-three hybridizing bands were found(results not shown) (0.5×SSC, 0.5% SDS, 65° C.). This suggests thatthere are at least two to three NRC1 orthologs present in the genome ofN. benthamiana that can be silenced upon inoculation with TRV:NRC1.

2.2 NRC1-Silenced Tomato is Affected in Cf-4-Mediated HR and DiseaseResistance

To investigate the function of NRC1 in HR-signaling and resistance to C.fulvum, the inventors performed VIGS in tomato, since this plant is theonly host for this fungus. Ten-day-old tomato seedlings wereagroinfiltrated with TRV:NRC1 and three weeks post infiltration RNA wasisolated from potentially silenced leaflets and analyzed by RT-PCR. TheNRC1 transcript levels varied in different TRV:NRC1-infected plants, butin most cases they were lower than in the TRV:00-infected plants,indicating that ‘knock-down’ of NRC1 expression had occurred (data notshown).

To exclude the possibility that the phenotype that we observe in tomatois caused by silencing of additional NB-LRR proteins, we also performedVIGS in tomato using a 360 bp fragment of NRC1 targeted to LRRs eight totwelve (TRV:NRC1-LRR), and a fragment consisting of 297 bp of the3′-untranslated region (UTR) of NRC1 (TRV:NRC1-UTR). With theseconstructs we tested whether NRC1 is required for Cf-4-mediated HR intomato by Avr4 protein injections in TRV:222-LRR andTRV:222-UTR-infected, Cf-4-containing tomato plants. Silencing of NRC1(using each of the three constructs) results in a mild phenotype as thetomato plants appeared somewhat smaller than the TRV:00- orTRV:Cf-4-infected plants (data not shown). As controls Avr4 protein wasinjected in TRV:00- and TRV:Cf-4-infected plants. In TRV:Cf-4-infectedplants the percentage of responding Avr4-injected sites was 52% (FIG.2), indicating a decreased HR due to silencing of Cf-4. In TRV:222-LRRand TRV:222-UTR-infected plants this percentage was similar (56% and48%, respectively) (FIG. 2), confirming the function of NRC1 inCf-4/Avr4-induced HR, also in tomato. Similar results were obtained uponVIGS of Nrc1 in Cf-9-containing tomato and subsequent injections ofapoplastic fluid containing Avr9 (not shown). For VIGS of Cf-9 inCf-9-containing tomato we used the TRV:Cf-4 construct, since the 404 bpCf-4 fragment codes for the highly conserved LRRs 15 to 21, enablingsilencing of both Cf-4 as well as the homologous Cf-9 resistance gene(Van der Hoom et al., 2001, supra).

Further, it was investigated whether NRC1 is also required for fullresistance of tomato to C. fulvum. Cf0 and Cf-4-plants were inoculatedwith TRV:00, TRV:Cf-4 and TRV:NRC1 and after three weeks silenced plantswere inoculated with a strain of C. fulvum expressing Avr4 and theβ-glucuronidase (GUS) gene, thereby allowing visualization of fungalgrowth. Two weeks post C. fulvum inoculation leaves were stained withX-gluc. In leaflets of Cf-4 plants infected with TRV:00 no growth of C.fulvum was detected, whereas in TRV:Cf-4-infected Cf-4 plants patches ofblue staining indicate compromised Cf-4-mediated resistance (not shown).Also in TRV:NRC1-infected plants small patches of blue staining indicateloss of full resistance against the fungus. Microscopical analysisrevealed intercellular growth of fungal hyphae in TRV:Cf-4- andTRV:NRC1-infected plants, but not in the TRV:00-infected control plants.All Cf0 plants displayed extensive colonization by C. fulvum, indicatingthat neither the TRV infection itself, nor VIGS using TRV:NRC1 affectsthe susceptibility of these plants to the fungus.

2.3 VIGS of NRC1 Affects the HR Induced by Different Matching R Gene/AvrGene Combinations

In addition to a decreased Cf-4/Avr4-induced HR upon VIGS using NRC1, itwas found that also the HR induced by the Inf1 elicitor of the oomycetepathogen Phytophthora infestans is decreased upon VIGS using NRC1 in N.benthamiana. To further investigate the specificity of NRC1 in defensesignaling, the inventors tested its requirement for the HR induced byadditional R/Avr combinations. As controls TRV:00 (empty vector) andTRV:SGT1 were included, since SGT1 is known to be required for the HRinduced by several R/Avr combinations (Peart et al., 2002, Proc. Natl.Acad. Sci. USA 99, 10865-10869).

Agroinfiltration of a mix of Cf-9 and Avr9 (Van der Hoom et al., 2000,supra), or a mix of LeEix2 and tvEix (Ron and Avni, 2004, Plant Cell 16,1604-1615) in TRV:NRC1-infected N. benthamiana resulted in a decreasedHR, whereas in the TRV:00-infected plants the HR developed normally. InTRV:SGT1-infected plants the HR was completely abolished, confirming theobservations of Peart et al. (2002, supra) (FIG. 3). Also AvrPto fromthe bacterial pathogen Pseudomonas syringae pv tomato and the geneencoding the coat protein (CP) of potato virus X (PVX) wereagroinfiltrated in TRV-infected N. benthamiana expressing the resistancegene Pto (Pedley and Martin, 2003, Annu. Rev. Phytopathol. 41, 215-243)and Rx (Bendahmane et al., 1999, Plant Cell 11, 781-791), respectively.In both cases plants infected with TRV:00 showed an HR, while the HR wasabolished in TRV:SGT1-infected plants. TRV:NRC1-infection resulted in aseverely suppressed Pto/AvrPto- as well as Rx/CP-induced HR, indicatingthat in N. benthamiana an NRC1 protein is required for HR signalingactivated by several R/Avr gene-for-gene combinations (FIG. 3).

To exclude the possibility that the compromised HR in TRV:NRC1-infectedN. benthamiana results from a decreased transformation efficiency byAgrobacterium, the inventors infiltrated TRV:00- and TRV:NRC1-infectedN. benthamiana:Cf-4 with Agrobacterium expressing the β-glucuronidase(GUS) gene (Van der Hoorn et al., 2000, supra). Three days postinfiltration a similar intensity of the blue staining in TRV:00- andTRV:NRC1-infected plants revealed that the transformation efficiency ofthe plants by Agrobacterium is not affected (data not shown). Inaddition, the TRV:NRC1-infected plants also showed a reduced HR uponinjection with Avr4 protein, while in TRV:00-infected plants a clear HRdeveloped within 2 days.

2.4 NRC1 Acts Downstream of EDS1 and Upstream of the MAPK Cascade in aCell Death Signaling Pathway

Since NRC1 is required not only for Cf-4/Avr4-induced HR, but also forHR induced by several additional R/Avr combinations, NRC1 appears to beinvolved in a common HR signaling pathway. A typical host response thatprecedes the initiation of the HR includes activation of MAPK cascades(Romeis et al., 2001, EMBO J. 20, 5556-5567; Del Pozo et al., 2004, EMBOJ. 23, 3072-3082; Pedley and Martin, 2005, Plant Biol. 8, 541-547).

To investigate the requirement of NRC1 for the HR initiated by MAPKs,epistasis experiments in N. benthamiana were performed. Plants wereinoculated with TRV:00, TRV:SGT1 and TRV:NRC1 and subsequentlyagroinfiltrated with genes encoding the kinase domain of LeMAPKKKα(LeMAPKKKα^(KD)) or constitutively active LeMEK2 (LeMEK2DD) (Yang etal., 2001, Proc. Natl. Acad. Sci. USA 98, 741-746; Del Pozo et al.,2004, EMBO J. 23, 3072-3082.). Two days post agroinfiltration expressionof the genes was induced by spraying the infiltrated leaves withestradiol. Transient expression of each of the genes results in an HR inTRV:00-infected plants, whereas in TRV:SGT1-infected plants the HR isdecreased (FIG. 4A). In TRV:NRC1-infected plants the HR caused by bothconstitutively active kinases is not affected (FIG. 4A).Agroinfiltration of the corresponding negative controls, LeMAPKKKα^(KD−)and wild-type LeMEK2 did not result in an HR in any of the TRV-infectedplants (data not shown). These results indicate that SGT1 is functionaldownstream of these MAPKs, whereas the MAPKs act either downstream orindependent of NRC1.

2.5 Transient Overexpression of NRC1 and Construction of aConstitutively Active NRC1 Protein

To further investigate which genes are required for HR signaling by theCC-NB-LRR protein the effect of overexpression of NRC1 was investigated.Therefore, the coding sequence (SEQ ID NO: 1) of the cDNA was fused tothe constitutive 35S promoter and inserted into a binary vector.Agroinfiltration of this construct in N. benthamiana did not result inan HR, whereas expression of a mix of NRC1 and the p19 silencinginhibitor (Voinnet et al., 2003, Plant J. 33, 949-956) did provoke anelicitor-independent HR (FIG. 4B). Agroinfiltration of a constructencoding a P-loop mutant of NRC1 (K191R) disrupting the P-loop motif,thereby affecting ATP hydrolysis (Tameling et al., 2002, Plant Cell 14,2929-2939), either with or without p19, did not result in an HR (FIG.4B).

The above described data indicated that post transcriptional genesilencing (PTGS) of the NRC1 gene may, therefore, prevent thedevelopment of an HR in NRC1 overexpressing tissue. Also, the disruptionof the P-loop motif results in a non-functional NRC1 protein.

Since mutations in the MHD motif of the NB-LRR resistance proteins Rx(D460V) (Bendahmane et al., 2002; Tameling et al., 2002) and I-2 (D495V)(Bendahmane et al., 2002, Plant J. 32, 195-204; Tameling et al., 2002,Plant Cell 14, 2929-2939; Van Bentem et al., 2005, Plant J. 43, 284-298)result in constitutive activity, the inventors generated a similarmutant of NRC1 (NRC1^(D481V)). Indeed, agroinfiltration of NRC1^(D481V)resulted in an elicitor-independent HR in leaves of N. benthamianawithin three days post infiltration and again no HR was observed uponagroinfiltration of the double mutant NRC1^(K191R/D481V) (FIG. 4B).Furthermore, no HR was induced upon expression of NRC1^(D481V) inSGT1-silenced plants (see below). These results indicate that theresponse induced upon agroinfiltration of NRC1^(D481V) is specificallydue to constitutive activity of the NRC1 protein and that NRC1 functionsin a signal transduction cascade leading to HR.

2.6 Epistatis Experiments Using a Constitutively Active NCR1 Protein

Epistasis experiments employing NRC1^(D481V) were performed to furtherinvestigate which genes are required for HR signaling by this protein,and thereby determine its putative position in an HR pathway. Inaddition to VIGS of genes known to be generally involved in HRsignaling, such as SGT1 and RAR1 (Required for Mla12 resistance)(Shirasu and Schulze-Lefert, 2003, Trends Plant Sci. 8, 252-258), N.benthamiana:Cf-4 was silenced for NDR1 (non race-specific diseaseresistance) (Century et al., 1995, Proc. Natl. Acad. Sci. USA 92,6597-6601), EDS1 (enhanced disease susceptibility) (Aarts et al., 1998,Proc. Natl. Acad. Sci. USA 95, 10306-10311) and MEK2 (a MAPKK) (Ekengrenet al., 2003, Plant J. 36, 905-917), and subsequently agroinfiltratedwith NRC1^(D481V) or Avr4. Furthermore, VIGS using TRV:00, TRV:Cf-4 andTRV:NRC1 was included as controls. A compromised NRC1^(D481V)- orAvr4-induced HR indicates ‘knock-down’ of a gene required forrespectively NRC1- or Cf-4/Avr4-induced HR signaling.

As expected, HR induced upon agroinfiltration of Avr4 was compromised inTRV:Cf-4- and TRV:NRC1-infected plants. Cf-4-mediated signaling alsorequires EDS1, as plants silenced for this gene displayed a less severeAvr4-induced HR. In addition, the inventors found a reduced HR uponagroinfiltration of Avr4 in plants silenced for MEK2, RAR1 and SGT1(FIG. 4C; light circles). The Avr4-induced HR is not compromised inTRV:00- and TRV:NDR1-infected plants (FIG. 4C; dark circles), indicatingthat NDR1 is not required for Cf-4-mediated signaling. Similarly,NRC1^(D481V)-induced HR was not compromised in TRV:00- andTRV:NDR1-infected plants, and also not in TRV:Cf-4-infected plants.Interestingly, in contrast to Avr4, NRC1^(D481V) still induces an HR inTRV:EDS1-infected plants, indicating that NRC1 is functional downstreamof EDS1 (FIG. 4C; dark circles). The NRC1^(D481V)-induced HR iscompromised in plants silenced for MEK2, showing that NRC1 requires theMAP kinase cascade for its signaling and can be positioned upstream ofthese kinases. VIGS of RAR1 and SGT1 also compromises D481V-induced HR,similar to the HR induced by Avr4 (FIG. 4C; light circles). Thus, NRC1is required for HR signaling initiated by Cf-4 and can be positionedupstream of the MAPK cascade and downstream of EDS1.

See FIG. 5 for a model of NRC1 mediated cell signaling.

Example 3 NRC1 Requirement for Mi-Mediated Resistance

In order to determine whether NRC1 is required for Mi-mediatedresistance against nematodes, white fly and aphids, a constitutivelyactive form of Mi (see U.S. Pat. No. 6,613,962 and EP0937155B1) isagroinfiltrated into NRC1 silenced plants. A decreased HR in NRC1silenced plants indicates that NRC1 is also required for Mi-mediated HRand that (over)expression of NRC1 can be used to generate transgenicplants having enhanced resistance against nematodes, white fly andaphids.

The above example is provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences,patents, and patent applications cited herein are hereby incorporated byreference.

1. A method for producing a transgenic plant having enhanced diseaseresistance compared to a non-transgenic control plant, said methodcomprising the steps of: a) transforming a plant or plant cell with anucleotide sequence encoding a protein comprising an amino acid sequenceat least 95% identical to SEQ ID NO:2, wherein the nucleotide sequenceis operably linked to a promoter active in plant cells, b) regeneratinga plant, wherein the regenerated plant has enhanced disease resistancecompared to a non-transgenic control plant.
 2. The method according toclaim 1, wherein said nucleotide sequence is integrated into the genomeof said plant.
 3. The method according to claim 1, further comprising c)screening the regenerated plant, or a plant derived therefrom by selfingor crossing, for resistance to one or more plant pathogens andidentifying a plant comprising enhanced resistance to one or more ofsaid plant pathogens.
 4. The method according to claim 1, wherein saidpromoter is a pathogen inducible promoter.
 5. The method according toclaim 1, wherein the protein comprises the amino acid sequence of SEQ IDNO:
 2. 6. The method according to claim 1, wherein the plant belongs tothe family Solanaceae.
 7. The method according to claim 6, wherein theplant is of the genus Solanum.
 8. A transgenic plant, plant cell, seedor fruit, obtainable by the method according to claim
 1. 9. A plant,plant cell, seed or fruit comprising a chimeric gene, the chimeric genecomprising a promoter active in plant cells operably linked to a nucleicacid encoding a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO:2.
 10. The plant of claim 9, wherein the plant isof the genus Solanum.
 11. The method according to claim 1, wherein theprotein comprises the amino acid sequence of SEQ ID NO:4.
 12. The plant,plant cell, seed or fruit of claim 9, wherein the protein comprises theamino acid sequence of SEQ ID NO:
 2. 13. The plant, plant cell, seed orfruit of claim 9, wherein the protein comprises the amino acid sequenceof SEQ ID NO:
 4. 14. A plant of claim
 9. 15. A plant cell of claim 9.16. A seed of claim
 9. 17. A fruit of claim
 9. 18. The plant, plantcell, seed or fruit of claim 9, wherein the plant belongs to the familySolanaceae.